Immunomodulatory and anti-tumor activities of native and heat denatured Abrus agglutinin

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Immunobiology 212 (2007) 589–599 www.elsevier.de/imbio

Immunomodulatory and anti-tumor activities of native and heat denatured Abrus agglutinin Dipanjan Ghosh, Tapas K Maiti Department of Biotechnology, Indian Institute of Technology, Kharagpur, West Bengal 721302, India Received 20 September 2006; received in revised form 9 February 2007; accepted 1 March 2007

Abstract Abrus agglutinin (AAG), a hetero tetrameric gal b (1–3) NAc gal specific lectin, is isolated from seeds of Abrus precatorius. In our previous studies we found that the protein could act as an immunomodulator and immunoadjuvant in native (NA) and heat denatured (HDA) conditions. An anticancer effect of the lectin is reported, but its mode of action is not clearly known. In the present study, the anti-tumor activity of AAG (NA, HDA) has been evaluated in a murine Dalton’s lymphoma (DL) ascites tumorogenic model. We found that treatment with both NA and HDA were able to decrease the tumor cell number in vivo and significantly increased median survival time. In vitro studies showed that AAG (NA, HDA) treatment of Dalton’s lymphoma ascites cells (DLAC) resulted in growth inhibition at the concentration of 1 mg/ml and above. Whereas, AAG (NA, HDA) at much lower concentrations (1 ng/ml) can stimulate peritoneal macrophage and spleen derived NK cells in vitro demonstrating cytotoxicity against DLAC. Cell cycle analysis showed an increased number of cells in Sub-G0/G1 phase for in vitro and in vivo treatments. In summary, AAG (NA, HDA) at non-toxic concentration was able to elicit anti-tumor effects in DL bearing mice by stimulating the innate immune system and Th1 type immunomodulation. r 2007 Elsevier GmbH. All rights reserved. Keywords: Abrus agglutinin; Lectin; Heat denatured; Dalton’s lymphoma; Immunomodulator; Immunoadjuvant; Anti-tumor

Introduction The host immune response to cancer is often very weak and immunosuppressive (Hadden, 2003; Ohm and Carbone, 2001). This situation prevents efficient eradication of tumor by the immune defense system (Das, 1995). The host response to cancer therapies may be stimulated by the administration of immunoadjuvants, which non-specifically activate the immune system (Ooi and Liu, 2000). Adjuvants with Th1 type of immunostimulation are most desirable for optimal host response Corresponding author. Tel.: +91 3222 83766; fax: +91 3222 55303.

E-mail address: [email protected] (T.K. Maiti). 0171-2985/$ - see front matter r 2007 Elsevier GmbH. All rights reserved. doi:10.1016/j.imbio.2007.03.005

in cancer immunotherapy. In this respect a variety of bacterial cell wall components, bacterial CpG oligonucleotide and peptides have been developed as immunoadjuvants for host immunostimulation against cancer (Dredge et al., 2002). In recent years plant lectins have been explored for immunomodulatory and immunoadjuvant properties for therapeutic purposes and certain lectins have evoked considerable interest as immunoadjuvants in cancer therapy (Beuth, 1997; Gabius, 2001). The effects of immunostimulatory lectins, e.g. phytohemagglutinin (PHA) (Wimer, 1990) and wheat germ lectin (WGA) (Ganguly and Das, 1991, 1994), on increasing mitogenic index of lymphocytes, stimulating cytotoxic effects of

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natural killer (NK) cells and macrophages, and upregulating the gene expression and secretion of cytokines and chemokines are well studied. Furthermore, soybean agglutinin (SBA) and WGA have been shown to reduce growth and progression of murine Dalton’s lymphoma (Ganguly and Das, 1991). The garlic lectin is also reported to induce apoptosis and have an anti-tumor effect on human tumor cells (Karasaki et al., 2001). The galactose specific lectin from Viscum alubum L. (VAA) is regarded as the principal immunostimulatory component of commercially available aqueous extract of mistletoe, historically employed in cancer therapy (Hajto et al., 1989). Another galactose specific lectin, Abrus agglutinin (AAG), isolated from Abrus precatorous L., is structurally similar to VAA and is considered to be an immunoadjuvant but is not widely studied. AAG, a ribosome inhibiting protein-II (RIP-II) family lectin, is a hetero-tetrameric glycoprotein of molecular weight 134 KD. It is composed of two A chains (N-glycosidase activity on 60S eukaryotic ribosome) and two B chains (galactose binding site) linked through a disulphide bridge (Liu et al., 2000; Olsnes et al., 1974). The lethal dose of AAG for 50% death (LD50) of mice was reported to be 5.0 mg/kg body weight and in vitro IC50 for protein synthesis inhibition is 469.0 mg/ml in mouse thymocytes (Hegde et al., 1991). In our previous works we had shown the Th1 type of immunomodulatory response by both native (NA) and heat denatured (HDA) AAG in mouse (Tripathi and Maiti, 2003a). Furthermore, NA and HDA were shown to stimulate peritoneal macrophage and NK cell activity in mouse splenocytes (Tripathi and Maiti, 2003b). Induction of tumor immunity by AAG in Meth-A fibrosarcoma and inhibition of the growth of sarcoma (S-180) in mouse were reported, but the mechanism of anti-tumor activity of AAG was not studied (Tung et al., 1979, 1981). The present study was designed to understand the effect of NA and HDA as immunoadjuvant to stimulate the innate immune system in a DL ascites tumorogenesis in Swiss albino mouse model.

Materials and methods Reagents and media 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), trypan blue, N-1-napthylethylenediamine dihydrochloride (NED), sulfanilamide and phosphoric acid were purchased from Loba chemicals (India). Lactic dehydrogenase (LDH) Cytotox assay kit was from Promega (USA). E. coli lipolysaccharide (LPS), propidium iodide (PI) and RNAase A were obtained from Sigma Chemical Co. (USA). Cytokine (IL-2, IL-4, IFN g) ELISA assay kits were from

Diaclone (France). Fetal bovine serum (FBS), RPMI1640 media, HBSS, Gey’s solution was obtained from Himedia (India). AAG was purified following the process used by Hegde et al. (1991) and isolated AAG was shown as a single band in polyacrylamide gel electrophoresis (data not shown). Heat denatured Abrus agglutinin (HDA) was prepared by heating AAG. Native Abrus agglutinin (NA) (5.0 ml, containing 1 mg/ml AAG in 10 mM phosphate buffer saline) was kept at 50 1C for 30 min and then kept immediately at 100 1C for 2 min in a water bath (Tripathi and Maiti, 2003a, b). It was made sure that there was no protein precipitation and protein concentration was estimated by the Lowry method (Lowry et al., 1951).

Mice Swiss albino male mice at 6–8 weeks of age and 2072 grams body weight were used throughout the study. Mice were housed in open top cages and maintained with proper food and water ad labitum. Room temperature was maintained 2272 1C. Light and dark cycle was 14/10 h.

Cell culture YAC-1 cell line was kindly provided by Dr. Ashok Khar, Centre for Cellular and Molecular Biology, Hyderabad, India. YAC-1 cells were maintained in RPMI-1640 media supplemented with 10% FBS. Mouse DL was maintained in vivo Swiss albino mice by serial intra-peritoneal (i.p.) transplantation into abdominal cavity. Dalton’s lymphoma ascites cells (DLAC) were collected aseptically from mouse peritoneal cavity and washed with RPMI-1640 media supplemented with 10% FBS, 100 IU/ml penicillin, 100 mg/ml streptomycin. The harvested DLAC were incubated in a Petri dish for 1 h at 37 1C in 5% CO2 atmosphere and non-adherent cells were cultured and used for in vitro experiments.

Induction of ascites in mice, treatment with AAG (NA, HDA) and survival of ascites bearing mice DLAC were harvested from the 7-days old ascites fluid, washed twice with RPMI-medium, suspended in 5.0 ml of RPMI media and counted by hemocytometer. About 500 ml of cell suspension (2  106/ml) was injected intra-peritoneally for the induction of ascites for control and treatment mice. Two treatment strategies (pre-treatment, and posttreatment) were followed in this study. Mice were injected 1, 3 and 5 days before the date of DLAC inoculation with NA, HDA (1 mg in 500 ml) for pretreatment and mice were treated 1, 3 and 5 days after

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mice treated with HDA 1, 3 and 5 days after initiation of ascites) and cell cycle analysis was performed.

a Pre-treatment Induction of tumor -5

-3

-1

591

Sacrificed on day 7

0

Isolation of peritoneal macrophages and nonadhering spleen cells

Days 1

3

5

b Post-treatment Fig. 1. Schedule for treatments. (a) Pre-treatment procedure follows treatment with NA or HDA (1 mg) 1, 3 and 5 days before the date of induction of tumor on day 0. (b) Posttreatment follows treatment with NA or HDA (1 mg) 1, 3 and 5 days after the date of induction of tumor. The mice were sacrificed on day 7 following induction of tumor.

DLAC inoculation for post-treatment as shown in Fig. 1. The day of DLAC inoculation was referred to as day zero. The injected mice were divided into two groups: DL control group (6 mice) and treatment group (6 mice for each treatment group). After 7 days of ascites development, mice were sacrificed by cervical dislocation and the anti-tumor effect was determined by counting the non-adhering viable DLAC numbers for treatment and control mice. Survival of DL bearing control and NA, HDA treated mice (15 in each group) was monitored by daily recording of mice mortality.

Effect of NA and HDA on DLAC in vitro and cell cycle analysis DLAC (1  105 cells/ml) were plated in 96 well flat bottom plates and incubated with various concentrations of NA and HDA (1 ng–100 mg/ml). All cultures were set up for 72 h at 37 1C in a humidified atmosphere at 5% CO2. In vitro effects of NA and HDA were checked by MTT assay (Mosmann, 1983). Cell cycle analysis of DLAC was performed after culturing the cells with NA and HDA (1 mg/ml) for 24 h. The cells were harvested, fixed in 70% ethanol and stored at 20 1C. These were washed with ice cold PBS (10 mM, pH 7.4), resuspended in 200 ml of PBS followed by incubation with 20 ml DNAase free RNAase (10 mg/ml) and 20 ml of DNA intercalating dye PI (1.0 mg/ml) at 37 1C for 1 h. Cell cycle analysis was performed by flow cytometry using Becton-Dickinson FACSCaliber flow cytometer and Cell Quest Pro software. DLAC were isolated from three groups of mice (Dalton’s lymphoma bearing mice, Dalton’s lymphoma bearing mice treated with NA 1, 3 and 5 days after initiation of ascites and Dalton’s lymphoma bearing

Swiss albino mice were sacrificed by cervical dislocation and peritoneal macrophages were isolated by intraperitoneal injection of 10 ml of ice-cold PBS (10 mM, pH 7.4). 1.0  106 macrophages were seeded in 12-well plates in RPMI complete medium, the non-adherent cells were removed after 2 h and the adherent macrophages were cultured for 24 h in RPMI complete medium. A single cell suspension of spleen from sacrificed mice was prepared under aseptic conditions by homogenization in Hank’s balanced salt solution (HBSS). The suspension was centrifuged to obtain a cell pellet. The contaminating RBC were removed by hemolytic Gey’s solution. After two washes in HBSS the cells were re suspended in complete RPMI medium. Cell concentration was adjusted to 1  106 cells/ml and seeded in 12-well plates in RPMI complete medium. The nonadherent cells were removed after 2 h and these were taken as a source of NK cells. The viability of nonadherent splenocytes (tested by trypan blue dye exclusion) was always over 90%.

Cytotoxic activity of macrophage and NK cell against DLAC In a 96 well tissue culture plate 90 ml of DLAC (1  105 cells/ml in RPMI complete media) were added to 90 ml of peritoneal macrophages of different concentrations so as to make the final ratios of effector macrophage: target DLAC (E:T) 10:1, 1:1 and 1:10. NA and HDA (20 ml) of different concentrations (1 ng/ml, 10 ng/ml and 100 ng/ml) were added to each well and incubated 24 h at 37 1C in a humidified atmosphere at 5% CO2. For control sets macrophages and DLAC, 90 ml of each cell was plated with 90 ml of complete media and 20 ml of media. The cytolysis activities of macrophages were measured by MTT assay (Mosmann, 1983). The cytolysis activity of macrophage was expressed as the percentage of tumor cytolysis as below (Moon et al., 1999): % Cytolysis ¼ ð1  fO:D:ðDalton’s lymphoma þ macrophageÞ  O:D:ðmacrophageÞg=fO:D: of Dalton’s lymphoma ðnontreatedÞgÞ 100.

Effector NK cells and target cells (DLAC) at ratios of 0.1, 1 and 10 were plated to a round bottom 96 well cell culture plate. NA and HDA were dispensed at different concentrations to the experimental wells. The plate was centrifuged at 250g for 4 min to ensure effecter and

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target cell contact. The cells were then incubated for 4 h in a humidified 37 1C, 5% CO2 incubator. After the incubation period the plate was again centrifuged at 250g for 4 min. The lactate dehydrogenase (LDH) concentration in the cell supernatant was measured by using Cytotox assay kit. The assay is based upon a coupled enzymatic assay involving the conversion of a tetrazolium salt 2-p-(iodophenyl)-3-(p-nitrophenyl)-5phenyltetrazolium chloride (INT), into a formazon product. The reaction was catalyzed by LDH released from cells and diaphorase present in the assay substrate mixture. Absorbance was read at 490 nm. The following formula was used to calculate % cytotoxicity: % Cytotoxicity ¼ ððexperimental  effector spontaneous  target spontaneousÞ=ðtarget maximum  target spontaneousÞÞ  100.

Macrophage induced nitric oxide production and NK cell activation in NA and HDA treated mice Swiss albino mice were divided into three groups including: (i) normal mice injected with PBS, (ii) normal mice injected with 1 mg of NA, and (iii) normal mice injected with 1 mg of HDA. 24 h after injection mice were sacrificed. Peritoneal macrophages and NK cells from splenocytes were isolated as stated earlier. Macrophages were seeded in 96 well plates (1.8  106 cells/well). These macrophages were activated by adding LPS (1 mg/ml) for nitric oxide production and cultured for 24 h at 37 1C in a humidified 5% CO2 incubator. Production of nitric oxide (NO) was estimated by measuring nitrite levels in the cell supernatant with the Griess reaction. Equal volumes of Griess reagent (1:1 of 0.1% N-1 naphthylethylenediamine in 5% phosphoric acid and 1% sulfanilamide in 5% phosphoric acid) and sample cell supernatant were incubated together at room temperature for 10 min and absorbance was read at 550 nm. NK cell activation was measured by cytotoxicity against NK sensitive YAC-1 cells following the same method described above (Tripathi and Maiti, 2005).

Splenocytes Proliferation Index (SPI) and cytokine induction from DL bearing mice Swiss albino mice were divided into four groups: (i) normal mice injected with PBS, (ii) Dalton’s lymphoma bearing mice treated with PBS, and Dalton’s lymphoma bearing mice treated with (iii) NA and (iv) HDA 1, 3 and 5 days after initiation of ascites. Mice were sacrificed on day 7 and splenocytes were isolated and concentration was adjusted to 1  106 cells/ml. The splenocytes (180 ml) were plated in 96 well flat bottom plates and incubated with 20 ml of various concentra-

tions (1 ng/ml–100 mg/ml) of HDA, NA. All cultures were set up in triplicate for 72 h at 37 1C in a humidified atmosphere of 5% CO2. Proliferation was checked by MTT assay method (Mosmann, 1983). Induction of cytokines (IL-2, IL-4 and IFN-g) by NA and HDA were performed by using splenocytes from: (i) DL bearing mice, and DL bearing mice treated with (ii) NA and (iii) HDA 1, 3 and 5 days after initiation of ascites. Mice were sacrificed on day 7 and splenocytes were prepared from the three groups of mice and cell concentration was adjusted to 1  106 cells/ml. The splenocytes (180 ml) were plated in 96 well flat bottom plates and each group of splenocytes were incubated with NA and HDA (each 100 ng/ml) for 72 h at 37 1C in a humidified atmosphere of 5% CO2. The culture supernatants were collected and used for the assay of cytokines (IL-2, IL-4 and IFN-g) by using ELISA kits from Diaclone following the manufacturer’s instructions.

Statistical analysis The data analysis was carried out using SPSS Ver. 12.0. The data were subjected to two-way ANOVA at 0.05% probability level of error. The mean values were separated by multiple comparisons following the HolmSidak method. All the pair-wise comparisons were made by student’s t-test. The survival functions of the three groups of mice were analyzed using the Kaplan–Meier method. P-value less than 0.05 was considered as significant.

Results Anti-tumor activity of NA, HDA in vivo The effects of pre- and post-treatment of AAG (NA, HDA) at a dose of 50 mg/kg body weight (injected three times) on Dalton’s lymphoma bearing mice were characterized by investigating the ascites non-adhering viable cell count after 7 days of ascites development. Pre-treatment with AAG (NA, HDA) was adopted to stimulate immune system of host prior to lymphoma inoculation, with a view to understand the role of preactivated immune cells on the growth restriction of cancer cells. The number of lymphoma cells in both preand post-treated groups was significantly less (Po0.001) in comparison to untreated control group (Fig. 2). Our results revealed that the post-treatment regimens (NA70.2%, HDA-54.39%) were more effective than pretreatment regimens (NA-40.1%, HDA-59.68%). This implied that continuous stimulation by immunoadjuvant AAG was needed. The anti-tumor activity of NA was found to be significantly different to that of HDA (Po0.05) in both regimens of treatment.

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593

120 Control Pre-treatment with NA Pre-treatment with HDA Post-treatment with NA Post-treatment with HDA

% of cell survived

100 80 60 40 20

Control

Pre-treatment

Post-treatment

Fig. 2. In vivo anti-tumor effects of NA and HDA in Dalton’s lymphoma bearing mouse ascites model. Pre-treatment involved three injections of NA or HDA (1 mg/500 ml) at 5, 3 and 1 days before the date of induction of ascites. At day 0, about 0.5 ml of 1  106 tumor cells were inoculated intra-peritoneally. Post-treatment involved the same dose of NA or HDA at 1, 3, 5 days after ascites induction. Mice were sacrificed on day 7 following induction. Data reported as the mean7S.D. for n ¼ 6 and compared against PBS control by using a Student’s t-test.

Survival of DL bearing mice after post-treatment with AAG (NA, HDA) We investigated the effect of post-treatment with AAG (NA, HDA) at a dose of 50 mg/kg body weight (injected three times) on the survival of DL bearing mice. The untreated DL bearing all mice died between 10 and 32 days. The median survival of mice in the control group, HDA group and NA group were 20, 24, and 23 days, respectively. Using Kaplan–Meier survival analysis, the differences in survival of both NA and HDA treated mice were found statistically significant compared to control DL bearing mice (Fig. 3, Table 1), but not between NA and HDA treated mice (P ¼ 0.3874). Accordingly, AAG (NA, HDA) treatment prolonged the life of DL bearing mice.

Fig. 3. Survival data to study NA and HDA post-treatment effects. Group 1, 2 and 3 indicate control, NA and HDA treated groups of mice, respectively. To elucidate treatment effects, NA or HDA (1 mg) were injected on 1, 3, 5 days after induction of ascites (15 mice in each group). Using Kaplan– Meier survival analysis, the median survival time increased from 20 days for Dalton’s lymphoma bearing mice treated with PBS (control) to 24 days in case of NA treated mice and 23 days in case of HDA treated mice. Table 1.

Survival outcomes of NA, HDA treatments

Group

Median survival7SEM (days)

P-value (vs. control)

Control NA HDA

2073.11 2475.03 2373.75

1 0.0385 0.0484

PBS NA HDA

120 100 % of cells survived

0

80

* *

60

* *

40

*

Cytotoxic effects of NA, HDA on DLAC in vitro

*

20

The result of cytotoxic activity of NA and HDA against DLAC is shown in Fig. 4. The percentage of growth inhibition by various concentrations of NA and HDA on DLAC was determined in comparison with viable cells of untreated controls. Data indicated that NA and HDA at lower concentration range (1 ng–100 ng/ml) had no inhibitory effect on DLAC growth. However, NA and HDA at higher concentration (4100 ng/ml) inhibited the growth of DLAC in a dose dependent manner. At the concentrations above 100 ng/ml, inhibition by NA was significantly more than

0

PBS

1

10 100 1000 10000 100000 Concentration (ng/ml)

Fig. 4. In vitro AAG (NA, HDA) induced cytotoxicity on DLAC. In a 96-well tissue-culture plate, 20 ml of NA/ HDA (1 ng–100 mg/ml) was added to 180 ml of Dalton’s lymphoma cells (105 cells/ml). MTT assay was performed after 72 h incubation at 37 1C and 5% CO2. Data reported as the mean7SD for n ¼ 12 and compared against PBS control by using a Student’s t-test. Po 0.05 were considered significant (*significant compared to PBS control).

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HDA at each concentration (P40.05, compared between NA and HDA values). The median inhibitory concentration (IC50) for NA and HDA was found to be 1 and 10 mg/ml, respectively.

PBS NA 1 ng/ml NA 10 ng/ml NA 100 ng/ml

PBS HDA 1ng/ml HDA 10 ng/ml HDA 100 ng/ml

120

In vitro DLAC cycle analysis The cell cycle analysis of in vitro NA/HDA (1 mg/ml for 24 h) treated DLAC resolved that the population in G0/G1 phase was increased from 35% to 49.2% (Po0.05) for NA and to 50.98% (Po0.05) for HDA. Total number of cells in S phase decreased from 24.0% to 5.0% and 8.0% for NA and HDA treatment, respectively (Fig. 5). This suggested that a portion of cells were arrested in G0/G1 phase after treatment with AAG (NA, HDA) and there is no significant difference between NA and HDA treatment.

% DLAC lysed

100 80 60 40 20 0 1:10

In vitro stimulation of macrophage and NK cell induced cytotoxicity on DLAC AAG (NA, HDA) was reported to stimulate macrophages to produce NO and TNFa and to activate NK cell mediated cytotoxicity on YAC1 as target cells in vitro. This prompted us to determine the in vitro activation of macrophages by NA and HDA and NK cell mediated DLAC (target cell) cytotoxicity. Our data indicated that both NA and HDA stimulated macrophages to induce cytotoxicity on DLAC (Fig. 6). Stimulation of macrophages by NA was maximum at 10:1 (E:T) ratio at the concentration of 1 ng/ml and

% of total cell

50

Sub G G0/G1 0/G1 G0/G1 G 0/G1 S G2/M G 2/M

40 30

1:01 10:01 1:10 1:01 Macrophage: Dalton's lymphoma

10:01

Fig. 6. Macrophage mediated DLAC killing in vitro. In a 96 well tissue culture plate Dalton’s lymphoma cells (105 cell/ml) were incubated with peritoneal macrophages of normal mice in ratios of 0.1, 1 and 10 such that the final volume is 180 ml. 20 ml of AAG (NA, HDA) was added as stimulant to each well. After 24 h MTT assay was performed. Data reported as the mean7SD for n ¼ 12 and compared against corresponding PBS control Po0.05 by using a Student’s t-test.

percentage Dalton’s lymphoma cell lysis was increased with increasing ratio of E:T cell (Po0.05). On the other hand, induction of macrophage stimulation was maximum only at1:1 ratio (E:T) at 10 ng/ml HDA. NK cell cytotoxicity on DLAC was determined at the concentrations 1–10 ng/ml NA and HDA at E:T ratios 0.1, 1 and 10 (Fig. 7). Maximum NK cell induced cytotoxicity was observed at the concentration of 1 ng/ml (16.25% for NA and 16.5% for HDA) at higher E:T ratios (10:1). NA induced NK cell activation was decreased with increasing concentration of NA, however, this was not observed for HDA.

20

Ex vivo and in vitro studies of macrophage and NK cell from AAG (NA, HDA) treated mice

10 0 CONTROL

NA treatment

HDA

Fig. 5. Cell cycle analysis of DLAC after treatment with AAG (NA, HDA) in vitro. Cell cycle analysis of Dalton’s lymphoma cells after culturing in vitro at 37 1C and 5% CO2 for 24 h in presence of NA and HDA (1 mg/ml). Analysis was done by flow cytometry using Becton-Dickinson FACSCaliber flow cytometer and Cell Quest software after cells being stained with PI. For this experiment, results are expressed as mean7SD for n ¼ 3.

The functional activity of AAG (NA/HDA at the concentration of 2 mg/ml) injected mice peritoneal macrophages was measured by LPS-induced NO production and that of spleen-derived NK cells was measured by YAC1 cell lysis in vitro. NA and HDA injected mice peritoneal macrophages exhibited higher amount of NO production (50 mM-NA, 35 mM-HDA) than that of PBS injected control (Po0.05) mice. Moreover, functional activation of NA injected mouse macrophages was significantly higher (Po0.05) than

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20 18

% DLAC lysed

16 14

PBS NA 1ng/ml NA 10ng/ml NA 100ng/ml HDA 1ng/ml HDA 10ng/ml HDA 100ng/ml

NO production in µM

22

12 10 8 6 4

65 60 55 50 45 40 35 30 25 20 15 10 5 0

2

595

Control mice NA injected mice HDA injected mice

PBS

0 1:10

1:01

10:01

Effector (NK cells): Target (DLAC)

Fig. 7. NK cell mediated DLAC killing in vitro. NK cells (non-adherent splenocytes taken as effector cells) were taken with Dalton’s lymphoma (target cells) in a 96-well tissueculture plate at effector: target ratios of 0.1, 1 and10. Cells were stimulated with 20 ml of AAG (NA, HDA) at different concentrations and incubated for 4 h at 37 1C. After incubation the plate was centrifuged and supernatant was taken for LDH assay. Data reported as the mean7SD for n ¼ 12 and compared against corresponding PBS control Po0.05 by using a Student’s t-test.

Fig. 8. In vivo macrophage activation by NA and HDA. Amount of NO produced was measured separately from in vitro LPS-activated peritoneal macrophages from normal mice and from the mice injected with NA and HDA (1 mg) 24 h before sacrifice. In each case, 180 ml of macrophage cell suspension was added in each well from one million cells/ml cell concentration. After 24 h NO was measured from cell supernatant by Griess reagent. Each value is represented as mean7SD for n ¼ 12, po0.05 as compared to control mice value.

Ex vivo and in vitro studies of SPI, cytokine secretion and cell cycle analysis of DLAC from DL bearing mice In tumor bearing mice immunosuppression prevails and it is worth investigating the effects of NA and HDA to prevent immunosuppression of DL bearing mice. SPI of AAG (NA, HDA) injected tumor bearing mice was determined after stimulating them in vitro by ConA (1, 10 ng/ml). We also measured the induction of cytokine (IL-2, IL-4 and IFN-g) production by splenocytes after in vitro stimulation with AAG (NA, HDA) 100 ng/ml following ELISA methods. SPIs of normal control (PBS treated), DL bearing mice: PBS treated, NA treated and HDA treated were 2.5, 1.5, 2.0 and 1.75, respectively, when splenocytes were stimulated by ConA (1 ng/ml), almost similar data were found for ConA at 10 ng/ml (Fig. 10). SPI data indicated that significant immunosuppression prevailed in ascites bearing mice (Po0.05

% YAC 1 cells lysed

25

that of HDA injected mouse (Fig. 8). NK cell activation data showed that in both NA and HDA injected mice, NK cells were significantly more effective (Po0.05) than those of control mice at lysing YAC1 cells at all the E:T cell ratios used. But the difference in NK cell activation between NA and HDA injected mice was not significant (Fig. 9).

LPS (1µg/ml)

control NA injected HDA injected

20 15 10 5 0 1:10

1:01

10:01

NK cells : YAC1 cells

Fig. 9. In vivo NK cells activation by NA and HDA. NK cell activity was measured by LDH assay. Effector cells (nonadherent splenocytes) were taken from mice, which were treated with 1 mg of NA or HDA separately 24 h before sacrifice. Target cells were YAC-1 murine cell line. Effector: target ratios were kept at 0.1, 1 and 10. Data are represented as mean7SD for n ¼ 12 and Po0.05 compared against corresponding PBS control value by using a Student’s t-test.

compared between control mice and DL bearing mice). Although there were increases in SPIs with AAG (NA, HDA) treatment in DL bearing mice in comparison to untreated DL bearing mice, indicating decrease of immunosuppression, these were not significant statistically (P40.05). Table 2 shows that splenocytes from the tumor bearing control mice (group-1) after activation with NA or HDA in vitro produced cytokines

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(IL-2, IL-4 and IFN-g) below the detection limit. Splenocytes from NA treated DL bearing mice (group2) and HDA treated DL bearing mice (group-3) produced enough IL-2 and IFN-g and production of IFN-g was less than that of IL-2. In all the cases, IL-4

control mice Cancer mice Cancer mice treated with NA Cancer mice treated with HDA

3.5 3.0

SPI

2.5 2.0

concentration was also found to be below the detection limit of the kit used. The cytokine induction data revealed that AAG (NA, HDA) treatment on DL bearing mice helped to generate Th 1 type of immune response. The cell cycle analysis of ex vivo DLAC from DL bearing mice revealed that treatment with AAG (NA and HDA) induced apoptosis (Fig. 11). The data indicated that the number of apoptotic cells increased significantly from 7.8% to 46.7% (Po0.001) and to35.98% (Po0.001) after NA and HDA treatment respectively. The cells in sub-G0/G1 phase also increased from 9.36% to 49.16% (Po0.001) and to 39.22% (Po0.001) for NA and HDA treatment, respectively.

1.5

Discussion

1.0 0.5 0.0

PBS

ConA 1ng/ml

ConA 10 ng/ml

CONCENTRATION

Fig. 10. Splenocyte Proliferation Index (SPI) of DL bearing mice. Splenocytes were isolated from four groups of mice: First group (mice without DL treated with PBS), and three groups of DL bearing mice (treatment with PBS, NA, HDA). Stimulation with ConA was done at doses of 1 and 10 ng/ml. Splenocyte Proliferations were assayed by MTT. Each value is represented as mean7SD for n ¼ 12, Po0.05 when compared with SPI of the control mice and DL bearing cancer mice; P40.05 compared with SPI of DL bearing cancer mice and the DL bearing cancer mice treated with NA or HDA. Table 2.

The aim of immunoadjuvant therapy is to stimulate the innate and adaptive immune systems to overcome the immunosuppressive situation in cancer patients alongside the conventional modes of cancer treatments, such as surgery, radiotherapy or chemotherapy. In the present study, our main objective was to evaluate the immunoadjuvant potential of galactose binding plant lectin Abrus agglutinin (NA, HDA) to restrict cancer development in a well-characterized Dalton’s lymphoma mouse ascites model. Interaction of cellular glycoconjugate with lectin in cell signaling induces cellular growth, cytotoxicity, and apoptosis; and anticancer potential of some plant lectins has been reported (Wimer, 1990, 2003; Ganguly and

Cytokine production by splenocytes from control (PBS treated) and AAG (NA, HDA) treated DL bearing mice

Groups

IL-2 (pg/ml)

IFN-g (pg/ml)

IL-4 (pg/ml)

Group-1 DL bearing control mice splenocyte (a) Control (PBS) (b) Stimulated with NA (c) Stimulated with HDA

ND ND ND

ND ND ND

ND ND ND

Group-2 NA treated DL bearing mice splenocyte (a) Control (PBS) (b) Stimulated with NA (c) Stimulated with HDA

8.571.27 43.3773.57* 40.1370.72*

6.1271.57 25.070.30* 18.072.80*

ND ND ND

Group-3 HDA treated DL bearing mice splenocyte (a) Control (PBS) (b) Stimulated with NA (c) Stimulated with HDA

9.272.17 34.3773.87* 27.5073.40*

5.571.12 12.572.1* 12.672.9*

ND ND ND

Splenocytes were stimulated by NA (100 ng/ml) and HDA (100 ng/ml) in vitro for 72 h and culture supernatants were used for measuring cytokines concentrations. The data presented are mean7 SD values of 12 replicates. Spleens from each mouse were collected after 7 days of induction of DL ascites. *Significant when compared with the corresponding PBS control.

ARTICLE IN PRESS D. Ghosh, T.K. Maiti / Immunobiology 212 (2007) 589–599

Counts

A

200

160

Sub G0 /G1 G0 /G1 S G2 /M

120

Apoptosis

= 9.54 = 42.48 = 14.86 = 33.02 = 8.18

200

400

80

40

0 0

600 FL2- H

800

1000

600 FL2 -H

800

1000

600 FL2 -H

800

1000

B 200 Sub G0 /G1 = 49.16 G0 /G1 = 19.58 = 11.76 S G2 /M = 21.02 Apoptosis = 46.70

Counts

160

120

80

40

0

C

0

400

200 Sub G0 /G1 = 39.22 G0 /G1 = 28.76 = 11.84 S G2 /M = 17.00 Apoptosis = 35.98

160

Counts

200

120

80

40

0 0

200

400

Fig. 11. Cell phase distribution of DLAC from DL bearing mice. Flow-cytometric analysis of cell phase distribution of DLAC isolated from 7 day ascites mice after the nuclei were labeled by PI preceding RNAase treatment using Cell Quest software. (A) DLAC from mice treated with PBS (control), (B) DLAC from mice treated with NA, and (C) DLAC from mice treated with HDA.

597

Das, 1994). Mistletoe extract containing VAA as a major constituent is frequently used as an alternative cancer therapeutic and immunomodulatory agent (Hajto et al., 2005). Studies on VAA conflict due to inherent toxicity and uncontrolled polarization of cytokine induction (Gabius, 2001). Strong mitogenic lectin Concanavalin A (Con A) was also reported to induce liver pathogenesis in mouse (Knolle et al., 1996; Leist and Wendel, 1996). Therefore, a plant lectin with low mitogenic activity and a less toxic effect may be a good choice as a potential immunomodulator in cancer therapy. The less studied lectin AAG (NA) is a weak mitogen and less toxic, these properties are reduced further in HDA due to heat inactivation of the galactose sugar binding pocket (Suryakala et al., 2000; Tripathi and Maiti, 2005). Previously, HDA was found to act as an immunomodulator in vitro (Tripathi and Maiti, 2003a, 2005) and that prompted us to use both NA, HDA to stimulate the immune system in order to restrain cancer growth both pre- and post-cancer cell transplantation and to improve the immunosuppressive situation in cancer-bearing mouse. In the present study, it was observed that both the pre- and post-treatment of AAG (NA, HDA) at non-toxic immunomodulatory concentration significantly decreased the lymphoma cell number in DL bearing mouse. The median-survival time of DL bearing mice treated with HDA and NA was increased significantly over that of control mice. The median inhibitory concentration (IC50) of Dalton’s lymphoma by NA and HDA in vitro was found to be around 1and 10 mg/ml, respectively, much higher than that of the immunomodulatory concentration. We propose that the observed growth restriction of lymphoma cells in AAG (NA, HDA) treated mice, is mediated by Th1 type immunomodulation and enhanced nitric oxide production by macrophages and NK cell mediated killing activity. This view is supported by our previous studies (Tripathi and Maiti, 2003a, b, 2005) and the present study on the activation of cell mediated killing of Dalton’s lymphoma cell in vitro at the non-toxic concentration of AAG (NA, HDA). Lectins from Tricholoma mongolicum were reported to inhibit tumor growth in sarcoma bearing C57BL/6 mice by enhanced nitric oxide production (Wang et al., 1997). Our study also revealed that the post-treatment was found to be more effective than pre-treatment indicating the necessity of constant stimulation of immune system by AAG (NA, HDA). In view of Dalton’s lymphoma growth restriction by AAG (NA, HDA) at toxic concentration in vitro and non-toxic immunomodulatory concentration in vivo, we performed cell growth phase analysis to understand the cell cycle checkpoint. The results revealed a significant apoptosis peak in the cell cycle in vivo and G0/G1 arrest in vitro. Adjuvant mediated induction of cancer cell apoptosis in vivo is most desirable (Schon and Schon,

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D. Ghosh, T.K. Maiti / Immunobiology 212 (2007) 589–599

2004). Whether AAG (NA, HDA) at very low concentration in the ascites environment induces apoptosis directly or is mediated by other mechanisms awaits further investigation. In vitro Dalton’s lymphoma cell cycle arrest by AAG (NA) at G0/G1 phase is possibly due to inhibition of protein synthesis, which in turn prevented entry of cells into S phase. It is known that cancer may induce immune suppression of the host leading to the inability of immune cells to restrict the proliferation of cancer cells in vivo (Bremers and Parmiani, 2000; Zou, 2005). In the case of cancer growth restriction, both innate and adaptive immune system of host is important. However, the innate immune system provides more rapid protection than the adaptive immune system (Valiante et al., 2003; Werling and Jungi, 2003). In the present study, our data indicates that innate immune cells were stimulated by AAG (NA, HDA) at low concentration (ng/ml) both in vitro and in vivo to produce reactive nitrogen intermediates and there was significant immunostimulation in DL-bearing immune-suppressed mice. Nitric oxide mediated tumor cytotoxicity is well reported. However, in many tumors nitric oxide is positively associated with tumor progression (Pollard, 2004). AAG (NA, HDA) treatment on ascites bearing mice upregulated Th1 cytokines (IL-2, IFN-g) and these may stimulate co-stimulatory molecules, subsequently activating adaptive immune cells. A Th1 cytokine environment may convert tumor-promoting macrophages to tumor destroying macrophages in the tumor microenvironment and activated macrophages demonstrate indisputable anti-tumor effects in various tumors (Bingle et al., 2002). In conclusion, our results indicate that AAG (NA, HDA) at non-toxic concentration exhibits therapeutic efficacy against murine Dalton’s lymphoma both in vitro and in vivo by stimulating innate immune cells. The immunostimulatory activities of NA are better than that of HDA, though NA is more toxic than HDA (IC50 of NA—1 mg/ml and HDA—10 mg/ml). Though in the present study we cannot show different immunomodulatory mechanisms between NA and HDA, our data demonstrate for the first time the anti-tumor effects of HDA in a mouse model. More investigations are needed to understand the mode of action of HDA on immune cells.

Acknowledgments The authors would like to acknowledge the Central Research Facility (CRF) of the Indian Institute of Technology, Kharagpur. The authors also would like to express thanks to Dr. S. K. Ghosh, Assistant Professor, Department of Biotechnology, Indian Institute of Technology, Kharagpur, for his help in FACS data

analysis and critical corrections of the manuscript and Mr. Sathya Sai Prasad for his help in statistical analysis.

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