Involvement of Chitosan-Cu Complex as Chemoprevention of Experimentally Carcinogenesis in Mice

Gamal Mohamed Fathy Edrees1, Maher Amer Ali Amer1*, HanaaAli Hassan1, Mostafa Al-Tonbary1

1Zoology Department, Faculty of Science, Mansoura University, Egypt

*Corresponding Author:Professor Maher Amer. Email: drmaher25@yahoo.com

ABSTRACT

Background: Chemoprevention is an extremely promising strategy for cancer prevention.Objective: The role of chitosan-cu complex (200 mg/kg) on oxidative stress induced by Erlich ascites carcinoma [EAC] (0.2X106 cells) in female mice was evaluated. Subjects and Methods: Eighty eight female Swiss albino mice weighing 23 ±3 g were divided into four equal groups, Control group (saline solution administrated orally at dose 200 mg/kg 16 days), Chitosan-Cu complex group (Chitosan-Cu complex administered orally at a dose of 200 mg/kg 16 days). Tumour induced group (Ehrlich ascites carcinoma cells were injected intra dermal the fourth day at dose 0.2×106 cells, Chitosan-Cu complex and tumour (Chitosan-Cu complex administered orally at adose of 200 mg/kg from the first day to the last day + Erlich ascites carcinoma. Results:Tumour size, argimase activity, α L-fucosidase and BL2 were significantly exceeded in EAC treated mice. In addition P53 level and Go/1 significantly decreased. These abnormalities are accompanied by increased the lipid peroxidation product (MDA) and decline in total antioxidant enzyme activity as well as GSH level,and mineral alteration. Conclusion: It can be concluded that the mice pretreated with chitosan-Cu complex then with EAC reveal marked protection as antitumor substance.

Key words:Chitosan Copper Complex, Ehrlich Ascites Carcinoma, Antioxidant

EdreesGMF, Amer MAA, Hassan HA, Al-Tonbary M. Involvement of Chitosan-Cu complex as chemoprevention of experimentally carcinogenesis in mice.Canad J ClinNutr 2014; 2 (2): 4-21

DOI:http://dx.doi.org/10.14206/canad.j.clin.nutr.2014.02.02

INTRODUCTION

Cancer continuous to represent the largest cause of mortality in the world and claims over 6 million lives every year (1). An extremely promising strategy for cancer prevention is chemoprevention, which is defined as the use of synthetic or natural agents (alone or combination) to prevent the development of cancer in humans (2).Chitosan (deacetylated derivative of chitin) is abundant, renewable, nontoxic and biodegradable carbohydrate polymers available in the exoskeletons of shellfish and insects. Chitosan has received much attention as a functional biopolymer for diverse applications, especially in pharmaceutics (3) and food (4) and cosmetics (5, 6).

Suzuki et al.,(7) revealed that the tumour inhibitory effect of Chitosan is  most likely  owing to their induction of lymphocyte cytokines and increasing T-cell proliferation, resulting in the anti-tumour mechanism of chito-oligosaccaridesand is enhanced by acquired immunity via increasing T-cell cytotoxicity and maintain T-cell activity (7).                          Maeda and Kimura (8), examined the anti-tumor effects of various low-molecular weight chitosans, such as water-soluble 21- or 46-kDa molecules with low viscosity, produced by enzymatic hydrolysis of over 650-kDa chitosan, which displayed decreased tumour growth and final tumour weight in sarcoma 180-bearing mice due to increase of natural killer cell activity.The goal of this study is to test whether chitosan copper complex inhibit the proliferation of tumor cells without damaging the normal cells, in experimentally solid tumour induced by Erlich ascites in female mice.

SUBJECTS AND METHODS

Materials:
Chitosan copper complex which contains 1 g Chitosan, 2 % acetic acid and 3.51 % copper was a gift from Prof. Dr. F Risha Physics Department, Faculty of Science, Mansoura University, Egypt. Ehrlish ascites carcinoma cells were purchased from National Institute of Cancer, Cairo, Egypt.

Animals and Diets:
Eighty eight female Swiss albino mice weighing 23±3 g were purchased from animal house facility of National Cancer Institute, Cairo, Egypt. The mice were housed in the animal facility of Zoology Department, Faculty of Science, Mansoura University, Egypt. Groups of six mice per cage in an air continued environment (25±2 ºC, 50-60% relative humidity). The animals were fed standard rodent diet and allowed water ad-libitum for a week for acclimatization and then were randomly assigned to 4 groups:  control group (saline solution administrated orally at dose 200 mg/kg 16 days), Chitosan-Cu complex group (Chitosan-Cu complex administrated orally at adose of 200 mg/kg 16 days).

Tumour induced group (Ehrlich ascites carcinoma cells were injected intradermal the fourth day at dose 0.2×106 cells, Chitosan-Cu complex and tumour (Chitosan-Cu complex administered orally at adose of 200 mg/kg from the first day to the last day + Erlichassites carcinoma cells where injected in the fourth day only at a dose 0.2×106 cells  Chitosan-Cu complex.After fourteen days tumor volumes were assessed, all the animals were sacrificed by cervical decapitation. Blood samples were collected in heparinized tubes and tumor and liver were immediately removed, weighed and stored at -20ºC.

Tumour growth measurements:
The development of subcutaneous tumours was assessed by a caliper by measuring the two dimensions of the palpable tumor and tumor volume was calculated using the equation V =   a b2 [0.4](9): Where, (V)  Is the volume;(a)  Is the major diameter;(b)  Is the minor diameter, and [0.4] is a constant.

Tumour markers
Arginase activity was determined by colorimetric kit, Marschet al., (10) (Biodiagnostic company, 29 Tahreer St., Dokki, Giza, Egypt). α L Fucosidase activity was determined using the method of El-Houseiniet al., (11) ( Biodiagnostic company , 29 Tahreer St., Dokki, Giza, Egypt ).

Cell cycle length
FACS caliber flow cytometer (Becton Dickinson, sunnyvale, CA, USA) at Children Hospital was used to analyze cell cycle. The average number of evaluated nuclei per specimen 20.000 and the number of nuclei scanned was 120 per second. DNA histogram derived from flow cytometry was obtained with a computer program for Dean and Jett mathematical analysis (12).

Oxidative stress biomarkers
Malondialdehyde (MDA) was assessed as described by Ohkawa, et al., (13) by using Colorimetric kit purchased from (Biodiagnostic Company, 29 Tahreer St., Dokki, Giza, Egypt). Total antioxidant capacity was assessed as described by Koracevic, et al., (14) using Colorimetric kit purchased from ( Biodiagnostic company , 29 Tahreer St., Dokki, Giza, Egypt).

Glutathione (GSH) was determined as described by Beutleret al., (15),  serum sodium by Trinder, (16), serum potassium as described by Sunderman and Sunderman(17),  serum calcium was assessed as described by Gindler and King (18), and serum iron was assessed as described by Dreux (19),  using Colorimetric kits from (Biodiagnostic Company, 29 Tahreer St., Dokki, Giza, Egypt).

Statistical analysis:
The results were analyzed by One Way ANOVA (analysis of variance) test and compared using Tukey’stest. The results were expressed as mean ± standard errors of mean (SEM). The values of P≤0.05 were considered statistically significant (20).

RESULTS

Administration of Chitosan–Cu complex, prior to EAC inoculation resulted a significant decrease (P<0.05) in the tumour volume as compared to EAC group (Table1). EAC implantation causes significant increase (P<0.05) in the serum arginase and α L-fucosidase activities as compared to control group. Administration of the chitosan -Cu complex prior to EAC inoculation produced significant amelioration (P<0.05) in the serum arginase ,α L-fucosidaseactivities and in Bcl2and proliferation in P53 as compared to that of EAC control group.

Table (2) showed that EAC induces significant increase (P<0.05) in synthetic phase (S %), post synthetic phase (G2/m) and apoptosis and significant decrease (P<0.05) in presynthetic phase (G0/1) as compared to that of the normal control group. Administration of Chitosan-Cu complex prior to EAC inoculation produced significant depletion (P<0.05) in  synthetic phase (S %) , post synthetic phase (G2/m) and apoptosis and significant increase in presynthetic phase (Go/1) as compared to that of EAC control group.

Table (3) showed that EAC induces significant increase (P<0.05) in tumour and hepatic malondialdehyde (MDA) concentration and significant decrease (P<0.05) in tumour and hepatic total antioxidant capacity and glutathione reduced concentration as compared to that of the normal control group. Administration of Chitosan-Cu complex prior to EAC inoculation produced significant decrease (P<0.05) in tumour and hepatic tissue of  MDA concentration and significant increase in the tumour and hepatic tissue of the total antioxidant capacity and glutathione reduced concentration  as compared to that of EAC group.

Table 4 showed that EAC induced significant decrease (P<0.05) in serum sodium and iron levels as compared to that of the normal control group. Significant increase (P<0.05) in serum potassium and calcium levels, on the other hand, administration of Chitosan-Cu complex prior to EAC inoculation produced significant increases (P<0.05) in the serum sodium and iron levels and significant decreases (P<0.05) in the serum potassium and calcium levels compared to that of EAC group.

DISCUSSION

Administration of Chitosan-Cu complex to the animals for three consecutive weeks did neither produce any significant difference in the body weight gain, nor did any toxic biocompatible, biodegradable effect or a favorable effect on the growth rate of the animal use, a view which is in agreements with Attia and Weiss (21).

In the present results, EAC inoculation induces significant increase in tumour size; may be due to rapidly growing Ehrlich tumour carcinoma with very progressive behavior (22). Our results are in good agreement with (23) that may lead to the neutrophilic inflammatory response which is essential to Ehrlich tumor controlling. However, the high influx of these cells promotes tumour development. This effect is probably related to angiogenesis and growing factors induced by inflammation that are necessary for tumour development. The Ehrlich ascetic tumour implantation induces a local inflammatory reaction, with increasing vascular permeability which may be due to the intense edema formation (24). The ascetic fluid is essential for tumour growing since it constitutes the direct nutritional source for tumor cells (25). Chitosan and its derivatives have been proved as antitumor agent and can suppress tumour growth and final tumor weight. It may be  due to increase of natural killer cell activity (26) who showed that, suppression of apoptosis (programmed cell death) by tumour promoting agents is an important mechanism in tumor promotion. The obtained decrease in tumor size in Chitosan-Cu complex treated group may probably through the decrease of ascites volume (8).

The present data showed that, that EAC inoculation induces significant increase in serum arginase activity as compared to that, of the normal control. This result agree with Erbas (27) who observed that in the solid tumour induced by Ehrlich ascites in female mice there was a significant  increase in serum arginase activity, the elevated arginase activity has been demonstrated in different carcinomas, indicating its relation to cancer. The administration of Chitosan-Cu complex showed significant decrease in serum arginase activity when compared with the mice loaded with solid tumour, may be attributed to the anti-inflammatory effects of Chitosan (28).

The present data showed that EAC inoculation induces significant increase in Serum α L-fucosidase (AFU) activity as compared to that of the normal control. This result agree with (29) who noted that, in the solid tumour induced by Ehrlich ascites in female mice there was a significant increase in AFU activity which indicate the appearance of cancer , as AFU activity is considered a tumour marker. The administration of Chitosan-Cu complex showed decrease in AFU activity compared with the mice loaded with solidtumor and are in line with (7) may be due to the interaction between chitosan and matrix metalloprotease II which decrease the invasive activity of tumour cells (30).

The present data showed that, in the solid tumor induced by Ehrlich ascites in female mice there was a significant  increase in Bcl2 % when compared with normal ones ,  this parameter is in a good agreement with the previous study of  (31) who noted  that, BcL2, is a proto-oncogene originally discovered in a follicular B-cell lymphoma .  The administration of Chitosan-Cu complex pre EAC showed decrease in Bcl2 % when compared with the mice loaded with solid tumour This result agree with (32)  and may be attributed to the cellular defense mechanism of Chitosan against ROS generation (33).

The reduced P53 in tumour loaded group was probably due to the  tumour suppressor function of P53 (35). It may also be attributed due to the activation of P53 leading to DNA damage as reported by (34), the administration of Chitosan-Cu complex showed significant increase in P53 percentage as compared with that in the mice loaded with solid tumour, possibly due to augmentation of the natural killer (NK) activity by low molecular weight , low viscosity of the used Chitosan-Cu complex (8).

Tumour cell extracted from the tumor of the animal group bearing tumor and treated with Chitosan-Cu complex showed an accumulation of cells in the S phase of the cell cycle. In this study, the population of cells in G0/1 phase decreased profoundly, while the increase in G2/m and S phase cells which indicated that Chitosan-Cu complex could delay or inhibit cell cycle progression through S phase. This delay may be attributed to the ability of Cu++ activity to bind with DNA arresting cell cycle and causes apoptosis , a view which run parallel with (36).

The role played by ROS in cellular processes including DNA damage , mitochondrial dysfunction , activation of signaling pathways and activation of transcription factors also leading to up regulate genes (37) this results are confirmed by the significant increase of MDA levels in the present work . In addition to the fact that, tumour cells are required excess copper and iron, that increases the intracellular ROS levels. ROS triggered by copper II present in Chitosan-Cu complex, which may be a key to cell death, regarding the significant reduction of serum iron concentration in animal bearing tumour administered with Chitosan-Cu complex, while iron is an absolute requirement for cell proliferation, energy metabolism and DNA synthesis.

Without iron, cells are unable to proceed from the G0/1 to the S phase of the cell cycle (38).The iron loss may also lead to decrease in hemoglobin synthesis and hence reduce oxygen uptake by the tumour cell leading to cell death.In addition, apoptosis is a crucial process related to a number of diseases and it induced by copper and its complexes (39). Also, the redox active properties of copper and its complexes can catalyze the formation of reactive oxygen species (ROS) which might induce the oxidative modification of cellular components, impair the intracellular redox balance, and for regulate the redox – related signaling pathways (40), as critical triggers in cell apoptotic pathways, (41).

The antioxidant activity of chitosan may have role in the decrease in DNA damage that contribute to degenerative change and cancer by reducing ROS in Chitosan-Cu complex administered group pre EAC inoculation, this explanation agree with Schulz et al., (42).  The present data showed that EAC inoculation induces significant increase in liver and tumour tissues malondealdehyde (MDA) level as compared to that of the normal control. The increased MDA level in tumor loaded group may be as a result of the excessive free radical production (2) .

The administration of chitosan-Cu complex showed significant reduction in hepatic and tumour tissues malondialdehyde (MDA) level when compared with the mice loaded with solid tumour, a result which may be attributed to the antioxidant activity of Chitosan-Cu complex, a view which in accordance with (33) or may be attributed to the complex – mediated cytotoxicity through generation of ROS, which in turn leads to cell death (43).

The present data showed that EAC inoculation induces significant reduction in hepatic and tumour tissues in total antioxidant capacity (TAC) as compared to that of the normal control, this result agrees a recent study(44) noted that, in the solid tumor induced by Ehrlich ascites in female mice there was a significant decrease in serum total antioxidant capacity (TAC), this decline may be attributed to the presence of ROS in tumor loaded group (37).

The administration of Chitosan-Cu complex showed amelioration in TAC in hepatic and tumour tissues when compared with the mice loaded with solid tumor, this amelioration may be resulted from the interaction of liposome with cell membrane exceeding the antioxidant capacity, a view which in accordance with (45).Also, it seems that the role of Cu++ ion in the catalytic cycle of SOD play a role in increasing the antioxidant status in chitosan-Cu complex treated group pre tumour induction (46).

The present results showed that, EAC inoculation induces significant reduction in hepatic and tumour tissues Glutathione reduced (GSH) level as compared to that of the normal control. This result agrees with Erbaset al., (27). They observed that, the solid tumour induced by Ehrlich ascites in female mice may be due to the presence of oxidative stress caused by ROS in tumour loaded mice (37). The administration of chitosan-Cu complex showed significant increase in hepatic glutathione (GSH), and tumour tissues compared with the mice loaded with solid tumour, this result agree with Drayton et al., (28) who noted that Chitosan and its derivatives has anti-inflammatory activity in biological medicine, this amelioration may be also resulted from the interaction of liposome with cell membrane exceeding the antioxidant capacity, a view which in accordance with Ngo et al., (45).

The present data showed that, EAC inoculation induces significant decrease in serum sodium concentration and significant increase in serum potassium as compared to that of the normal control. These  results   may be due to pathological changes and biochemical alternation induced after EAC inoculation,(47) The administration of chitosan-Cu complex showed significant amelioration in serum sodium and potassium concentration when compared with the mice loaded with solid tumour. The resulted amelioration in Chitosan-Cu complex treated group pre-tumour loaded may be attributed to the chelation activity of potassium ion by Chitosan-Cu complex (48).

A significant increase in calcium concentration and a significant decrease in iron concentration were also observed in the solid tumor induced by Ehrlich ascites in female mice when compared with normal ones. The increase in calcium may be due to higher secretion of parathyroid hormones (49) and the decrease in iron may be due to higher cellular iron uptake where iron overload associated with cancer risk (50).The administration of Chitosan-Cu complex showed significant decrease in serum calcium concentration and a significant increase in iron concentration when compared with that in the mice loaded with solid tumour,  the decrease of serum calcium concentration may be attributed to the excretion of calcium with urine where (51)showed that chitosan accelerating the excretion of urinary calcium, the amelioration in iron concentration may be through decrease in transferrin receptor in tumour cells through redox regulation  as previously reported (52).

In conclusion, the present study provided obvious evidence on the beneficial effects of Chitosan-Cu complex in reducing tumour growth and counteracting the metabolic disorders associated with female mice inoculated with solid tumour model and may offer novel approaches to cancer therapy.

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Table 1: Protective effect of chitosan copper complex on serum arginase and alpha L fucosidase activities as a tumor marker as well as Bcl2 % as antiapoptoticmarker, P53 as apoptotic and oncogenic markers in tumortissue in control and different treated mice groups

Significance

Chitosan-Cu+tumor

Tumor

Chitosan copper complex

Control

Animal groups

P<0.05*

0.2± 0.04

0.5±0.04

0.0±0.0

0.0±0.0

Tumor size

( cm3 )

P<0.05*

140.4 ±2.81

166.6±5.05

134.3±3.36

136.8±1.47

Arginase

(U/L)

P<0.05*

7.9 ±0.16

10.4 ±0.69

1.8 ±0.15

2.0 ±0.14

α L-fucosidase(U/L)

P<0.05*

19.1 ±0.16

28.9 ±0.22

8.6 ±0.61

10.7±0.37

Bcl2

(%)

P<0.05*

34.6 ±1.36

26.7 ±0.89

51.9 ±2.47

73.6±0.80

P53

(%)

Results are means ± SEM, *significantly different

Table 2: The effect of chitosan -Cu complex on DNA cycle

Significance

Chitosan-Cu+tumor

Tumor

Chitosan copper complex

Control

Animal group

P<0.05*

61.8±0.144

35.1±3.69

75.9±1.74

86.2±0.56

G0/1

P<0.05*

9.8±1.15

14.1±0.21

4.4±0.33

2.3±0.13

Synthetic Phase (S %)

 

P<0.05*

30.0±1.53

50.8±1.38

20.0±0.79

11.57±0.69

G2/m

P<0.05*

14.4±1.07

39.2±0.86

8.9±0.52

4.1±0.28

Apoptosis

(%)

Results are means ± SEM, *significantly different

Table 3: The antioxidant effect of Chitosan-Cu complex

Significance

Chitosan-Cu+tumor

Tumor

Chitosan copper complex

Control

Animal group

P<0.05*

232.3±5.91

446.2±6.88

116.5±2.96

170.2±4.58

MDA (tissue tumor) (nmol / gtissue )

P<0.05*

209.3 ±1.76

148.2 ±4.45

316.4 ±1.77

290.0±6.04

TAC (tissue tumor)(mM /1000 g tissue)

 

P<0.05*

67.0 ± 1.94

46.7± 1.09

93.8 ±1.08

83.1±3.23

GSH (tissue tumor)

(mg / g tissue)

P<0.05*

86.4 ± 3.91

156.8±5.13

48.3± 1.80

68.5±1.48

MDA (liver)

(nmol / g tissue )

 

P<0.05*

219.3 ±2.33

176.5±0.96

288.8±1.35

292.5±2.26

TAC (liver)

(mM /1000 g tissue)

P<0.05*

34.7 ± 1.50

22.2 ±1.53

46.0 ±2.20

44.9±2.67

GSH (liver)

(mg / g tissue)

Malondialdehyde (MDA),Total Antioxidant Capacity (TAC), and Glutathione Reduced (GSH)

Results are means ± SEM, *significantly different

Table 4: Effect of Chitosan-Cu complex on serum sodium,potassium,calcium, iron concentrations

Significance

Chitosan-Cu+tumour

Tumor

Chitosan copper complex

Control

Animal group

P<0.05*

164.3±3.16

118.8±1.68

161.4±7

169.1±5.84

Sodium

(mmol / L )

P<0.05*

4.5±0.16

6.8±0.39

4.6±0.11

3.6±0.16

Potassium

(mmol / L )

P<0.05*

6.9±0.32

7.6±0.12

5.2±0.24

6 ±0.28

Calcium

(μg /dL)

P<0.05*

40.7±1.39

30.6±1.28

68.4±0.41

68.6±1.17

Iron

(μg /dL)

Results are means ± SEM, *significantly different

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