Download Antibacterial and antitumor effects of bis-(4- bromobenzaldehyde

Advances in Environmental Biology, 10(12) December 2016, Pages: 159-170
AENSI Journals
Advances in Environmental Biology
ISSN-1995-0756
EISSN-1998-1066
Journal home page: http://www.aensiweb.com/AEB/
Antibacterial and antitumor effects of
bromobenzaldehyde-4-iminacetophenone)
tetraaquocobalt(ΙΙ) sulphate complex
bis-(4-
1,2Amani
F. H. Noureldeen, 1Safaa Y. Qusti, 1Wafa A. Alamoudi, 1Al-Anoud I. Rawas and 3Ramadan M
Ramadan
1
Biochemistry Department, Faculty of Science, King Abdulaziz University, Jeddah, Saudi Arabia.
Biochemistry Department, Faculty of Science, Ain Shams University, Cairo, Egypt.
3
Chemistry Department, Faculty of Science, Ain Shams University, Cairo, Egypt.
2
Address For Correspondence:
Amani F. H. Noureldeen, Biochemistry Department, Faculty of Science, King Abdulaziz University, Jeddah, Saudi Arabia
This work is licensed under the Creative Commons Attribution International License (CC BY).
http://creativecommons.org/licenses/by/4.0/
Received 12 August 2016; Accepted 17 December 2016; Available online 31 December 2016
ABSTRACT
The present study aimed to evaluate the antibacterial and anticancer activities of bis-(4-bromobenzaldehyde-4-iminacetophenone)
tetraaquocobalt(ΙΙ) sulphate complex (BBIA-Co). The antibacterial activity of the synthesized complex was tested against two bacterial
strains; Staphylococcus aureus and Escherchia coli. HepG2, A549 and PC3 carcinoma cell lines were used for evaluating in vitro
cytotoxicity of the complex. For the in vivo study, 125 mice were classified equally into 5 groups: normal, EAC bearing mice, vehicle,
BBIA-Co (healthy mice) and treated (mice inoculated with EAC cells) groups. Results indicated that the BBIA-Co complex has the capacity
to inhibit the metabolic growth of the tested microorganisms. In vitro study demonstrated medium cytotoxicity against tested cell lines with
IC50 values equivalent to 25.0 µg/ml, 17.5 µg/ ml and 21.7 µg/ ml for HepG2, A549 and PC3 respectively. In vivo study indicated that
BBIA-Co complex treatment had successfully increased mean survival time which in turn resulted in prolonged the life span of EAC-tumor
bearing mice (77%). The complex has effectively reduced the accumulated ascites fluid and decreased cell viability. The hematological
parameters and other biochemical parameters were significantly restored towards normal level post BBIA-Co-treatment, indicating the
effectiveness of the complex as an anticancer agent.
KEYWORDS: Cobalt complexes; Biological activity; Ehrlich tumors; In vitro cytotoxicity; In vivo cytotoxicity.
INTRODUCTION
Cancer or malignant neoplasm is a class of diseases in which a group of cells display uncontrolled growth,
invasion and even sometimes metastasis [16,55]. It continues as a serious public health problem throughout the
world as the most feared diagnosis. It is the second leading cause of human death after cardiovascular diseases
in developing and undeveloped countries [7]. Treatment for cancer primarily includes surgery and
chemotherapy, but the curative effects of the existing chemotherapeutic drugs are not good enough and they
have plentiful side effects. The development of more effective drugs for treating patients with cancer has been a
main attempt over the past 50 years [25,27,4].
Researches have shown significant progress in the utilization of metal complexes as drugs to treat several
human diseases like carcinomas, lymphomas, infection control, anti-inflammatory, diabetes and neurological
disorders. Schiff base ligands readily coordinate with a range of metal ions yielding stable complexes which
exhibit interesting physical, chemical, biological and catalytical properties [44]. Schiff base metal complexes
formed with metals like copper, gold, gallium, germanium, tin, ruthenium, iridium have shown significant
Copyright © 2016 by authors and Copyright, American-Eurasian Network for Scientific Information (AENSI Publication).
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antitumor activity in animals [45]. There has been considerable interest in the chemistry of the metal complexes
of Schiff bases containing O, N and S donor [52]. Despite the success of cisplatin; one of the most active and
clinically useful agent used in the treatment of cancer, however, it lacks selectivity for tumor tissue, which leads
to severe side effects. Various tumor cell lines are now growing resistance to cisplatin [51]. For this reason, new
research area are currently directed towards the development of more effective drugs with minimum side effects
for treating patients with cancer.
Recently, Ramadan et al., [46] reported the existence of medium in vitro cytotoxicity for a novel cobalt
complex; bis-(4-bromobenzaldehyde-4-iminacetophenone) tetraaquocobalt(ΙΙ) sulphate against human liver
carcinoma cell line (HepG2). The present study aimed to evaluate the biological activity as well as in vitro and
in vivo cytotoxicity of that reported complex (BBIA-Co), Scheme 1.
Scheme 1: Structure of the [Co(BBIA)2(H2O)4] SO4 complex
MATERIALS AND METHODS
a) Preparation of bis-(4-bromobenzaldehyde-4-iminacetophenone )tetraaquocobalt(II) sulphate:
The complex (BBIA-Co) under investigation was prepared according to the method described by Ramadan
et al., [46].
b) Biological activity:
Antibacterial activity of the synthesized complex was tested using paper diffusion disc procedure. The
activity was screened against two bacterial strains; Staphylococcus aureus as a gram-positive bacteria and
Escherchia coli as a gram-negative bacteria. Comparison of the obtained data was carried out with the two
standards: Amikacin and ciprofloxacin antibacterial agents. The nutrient agar medium; containing peptone, beef
extract, NaCl and agar-agar bacteriological grade B&V (purchased from the agar company; Italy); and 5mm
diameter paper discs of Whatman No.1 were used. The test compound was dissolved in DMSO (Sigma
Chemical Company) in 0.1-0.4% concentrations. The paper discs were soaked in different solutions of the
compound, dried and placed in the Petri plates (9 cm diameter) previously seeded with the test organisms. The
plates were incubated for 24-30 h at 27 ±1°C and diameter of the inhibition zone (mm) was measured around
each disc.
c) In vitro study:
Liver (HepG2), lung (A549) and prostate (PC3) carcinoma cell lines were used for in vitro screening
experiments of the tested complexes. The cancer cells were obtained frozen in liquid nitrogen (-180 °C) from
the American Type Culture Collection, Manassas, UA, USA.The sensitivity of the human tumor cell lines to
thymoquinone was determined by the sulforhodamine B (SRB) assay. This method was carried out according to
that described by Skchan et al. Cells were used when 90% confluence was reached in T25 flasks. Adherent cell
lines were harvested with 0.025% trypsin (Sigma Chemical Co., St. Louis, Mo., USA). Viability was
determined by trypan blue exclusion using the inverted microscope. RPMI-1640 medium (Sigma Chemical Co.,
St. Louis, Mo, and USA) was used for culturing and maintenance of the human tumor cell lines. Cells were
seeded in 96-wells microtiter plates at a concentration of 5×10 4-105 cells/well in a fresh medium and was left to
attach to the plates for 24 h. Growth inhibition of cells was calculated spectrophotometrically using a standard
method with the protein-binding dye SRB, Monks et al. [38]. The optical density (OD) of each well was
measured at 564 nm with an ELIZA microplate reader (Meter Tech. R 960, USA). The percentage of cell
survival was calculated as follows:
Survival fraction = O.D of treated cells / O.D of control cells.
The IC50 values are the concentrations of thymoquinone required to produce 50% inhibition of cell growth.
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d) In vivo study:
Materials:
EAC cells:
Cells were obtained from American Type Tissue Culture Collection, Manassas, VA, USA. Cells were
maintained in vivo in Swiss albino mice by intraperitoneal transplantation of 2 x10 6 cells/mouse after every 8
days [47].
Vehicle:
The BBIA-Co complex was freshly dissolved, directly before use, in a vehicle containing dimethyl
sulfoxide (DMSO) and distilled H2O (4:1 v/v).
Animals Management and Groups:
125 Male Swiss albino mice (8-10 weeks of age, 30-33 g body weight) were obtained from King Fahd
Medical Research Center, King Abudlaziz University, Jeddah, Saudi Arabia. Animals were kept for one week
acclimatization period under controlled conditions of temperature (23-25 oC), humidity (50-55%) and light/dark
cycle (12h L/12h D). Animals had free access to water and standard diet throughout the experiment. Mice were
divided randomly and equally into five main groups:
- Normal group: healthy animals belonging to this group were ip. injected with 0.2 ml saline, 3
times/week for 2 weeks.
- EAC group: animals in this group were ip. inoculated with 2x106 Ehrlich ascites carcinoma
cells/mouse and were monitored for 14 days.
- Vehicle group: mice were ip. injected with 0.2 ml of the vehicle used to dissolve BBIA-Co complex
(DEMSO:H2O, 4:1 v/v)
- BBIA-Co group: in this group, healthy animals were ip. injected with 0.2 ml of freshly dissolved Co
complex at a final dose equivalent to 40 mg of BBIA-Co/kg body weight (representing 10% of the lethal dose
for BBIA-Co), 3 times/week for 2 weeks.
- Treated group: animals in this group were i.p. inoculated with EAC cells (2x106 cells/mouse) followed,
after 24h, by injection of 40 mg BBIA-Co complex/ Kg body weight dissolved in 0.2 ml vehicle, 3 times/week
for 2 weeks.
Blood Samples:
Animals were monitored regularly for alterations in body weight, for the development of any signs of
toxicity and mortality. After the last dose, five mice from each group were left for survival study, while the rest
of animals were overnight fasted. Blood samples withdrawn from four mice in equal amount were pooled on a
clean tube; resulting in five samples in each group to obtain sufficient quantity of serum for determination of
biochemical parameters.
Methods:
Cytotoxicity parameters:
a. Food intake:
The amount of food intake/mouse/weak was calculated using the formula described by Anand et al., [6]:
Feed consumed by 5 animals/cage/week =
Total quantity of feed offered during that week (gm) - Feed left over on last day of week (gm)
Feed consumed by individual animal/week =
Feed consumed by 5 animals/cage/week ÷ number of mice in the cage
b. The percent change in body weight (B.Wt):
Body weight was registered for each mouse at the begging of experiment (zero day) and at day 15 (day of
animal sacrifice). The Percent of change in body weight was calculated using the formula described by Kuttan et
al., [32]:
Percentage change in B.Wt = (W2 - W1) / W1 x 100
Where W1 is the body weight of each mouse at the start of experiment and W 2 is the body weight of animal
at the end of observation.
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c. Mean Survival time (MST) and Life Span (%LS):
Animal survival time, for each group, was recorded and was expressed as mean survival time (days). Both
MST and LS (%) were calculated using the equations described by Mazumder et al., [35] and Gupta et al., [23]:
MST = (Day of 1st death + Day of last death) / 2
% LS = [(MST of treated group/ MST of control) - 1] × 100
d. Ascetic Fluid Collection:
At the end of the experiment and before blood collection, ascetic fluid (if present) was collected and its
volume was measured. EAC cells were harvested and cells viability was tested by trypan blue exclusion test
using hemocytometer. The total number of cells/ml was calculated using following equation:
Cell count = (number of cells x dilution) / (area x thickness of liquid film)
Hematological parameters:
Blood was obtained from retro-orbital sinus of the mice under light ether anesthesia using heparinized
micro-capillaries. Red blood cells (RBCS) and white blood cells (WBCS) were counted by auto-hematology
analyzer (BC-2800Vet, Mindray, China). Hemoglobin was measured photometricallly by fully-automated
instruments.
Biochemical Assays:
Separated sera were used for determination of liver and kidney functions by measuring: activities of alanine
amino transferase (ALT), aspartate amino transferase (AST) and alkaline phosphatase (ALP); in addition to
levels of: total proteins, albumin, globulin, urea, creatinine and uric acid. Determination of serum glucose and
lipid profile (triglycerides, cholesterol and very low density lipoproteins) was also carried out. All the
biochemical parameters were determined using specific kit for each parameter.
Statistical Analysis:
Statistical analysis was performed using SPSS 24.0 for windows (SPSS Inc, USA). Descriptive statistics
were shown as mean ± standard error of the mean. Kruskal-Wallis test was performed for comparing more than
two groups of ordinal, non-parametric data to determine if they were statistically different. Independent
samples’ Mann-Whitney (U) test was used as a post hoc test to compare the medians of two groups of ordinal,
non-parametric data to determine if they were statistically different. Correlation coefficient (r) was used to
estimate the effect of size that describe the proportion of total variability attributable to a Variable & used
Cohen’s effect size estimates to interpret the meaning of the (r) score. P value < 0.05 was considered
statistically significant.
Result:
Biological activity:
Results obtained from testing the potential activity of BBIA-Co against Staphylococcus aureus and
Escherichia coli indicated the ability of the complex to inhibit the growth of both bacterial stains, although the
antibacterial activity was lower than the antibacterial standards (Fig. 1).
In vitro study:
Results indicated that BBIA-Co complex exhibited medium cytotoxicity against the tested cell lines. The
IC50 value for liver carcinoma cell lines (HepG2) was equivalent to 25 µg/ml, while the IC 50 values for both
lung (A549) and prostate (PC3) carcinoma cell lines were 17.5 µg/ml and 21.7 µg/ml, respectively, as indicated
in Fig. 2.
In vivo study:
Cytotoxicity parameters:
Food Intake:
Results indicated significant increase in food intake by mice bearing EAC tumor, both at 1 st and 2nd week,
compared to normal mice. Same trend was noted for BBIA-Co treated EAC inoculated mice (Table 1).
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Fig. 1: Biological activity of BBIA-Co complex
Fig. 2: In vitro cytotoxicity of BBIA-Co complex against: liver (A), lung (B) and prostate (C) Carcinoma cell
lines.
Table 1: The food intake (g/mouse) for the studied groups
Groups
Normal
EAC
Vehicle
Parameter
1st week
11.5±0.29
14.4±0.19
10.6±0.16
*p value
-0.001
0.01
**p value
--0.001
2nd week
10.1±0.43
15.8±0.38
9.3±0.27
*p value
-0.001
NS
**p value
--0.001
*
p: p value with respect to normal group, **p: p value with respect to EAC group.
p value ≤ 0.05 is significant, NS: non-significant.
BBIA-Co
Treated
12.3±0.54
NS
0.04
13.4±0.27
0.001
0.001
14.4±0.30
0.001
NS
15.0±0.11
0.001
0.001
Change in Body Weight:
The percent change in body weight of EAC group, between day zero and day 15, was significantly higher
than the matched mean values in control and other groups. No significant difference between the percent change
in body weight was noticed between BBIA-Co treated mice and control healthy animals (Fig. 3). It is worth to
mention that at the beginning of the experiment all animal groups were matched for body weight.
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Mean Survival Time (MST), Life Span (%LS):
MST and % LS were calculated for both drug (BBIA-Co) and treated groups. Normal mice group was used
as a control for BBIA- Co group, while EAC group was used as a control for treated group. It is worth noting to
indicate that the survival of mice in both normal and vehicle groups were monitored for 120 days and no death
in both groups was noted. Treatment with 40 mg Co-complex /kg B.Wt following EAC inoculation improved
the mean survival time of mice which in turn resulted in prolonged life span. Normal mice injected with BBIACo complex showed reduction in their MST resulting in 21.5 % decreased life span (Table 2).
Fig. 3: The percent change in body weight.
p value with respect to normal group. Numbers inside the coulmn indicate standard error.
p value ≤ 0.05 is significant; NS: non-significant.
Ascetic Fluid Volume and EAC viability:
Untreated EAC bearing mice had ascetic fluid volume significantly higher than EAC bearing mice treated
with BBIA-Co. Moreover, treatment had significantly reduced the number of viable cells (13 %), resulting in
increased percent of non-viable cells (Table 2).
Hematological Profile:
Results indicated that inoculation of EAC cells to mice resulted in significant reduction in Hb content and
RBCs count, with elevated WBCs compared to normal mice group. Significant improvement in hematological
parameter, (Hb, RBCs and WBCs) was obvious by treating EAC inoculated mice with Co complex, reaching
WBCs mean value comparable to normal (Table 3).
Table 2: Mean survival time (MST), life span (LS), ascetic fluid volume and cell viability.
Groups
Normal
EAC
Parameters
MST (days)
120
26.5
ILS (%)
Ascetic fluid volume (ml)
12.8 ±1.70
*
p
Viable cells
8.6 ± 1.54
(×108 cells/mouse)
*
p
Non-viable cells
1.1 ±0.26
(×108 cells/mouse)
*
p
% of viable cells
88.8 ±0.97
*
p
% of non-viable cells
11.0 ±0.99
*
p
- MST and ILS were calculated for BBIA-Co group using normal group as a control group.
- MST and ILS were calculated for BBIA-Co treated group using EAC group as a control group.
*
p: p value with respect to EAC group. p value ≤ 0.05 is significant.
Vehicle
BBIA-Co
Treated
120
-
94
- 21.5
-
-
-
47
76.6
1.7 ± 0.27
0.0001
2.7 ± 0.47
-
-
0.0001
11.8 ± 1.81
-
-
-
-
0.0001
13.3 ± 1.93
0.0001
86.7 ± 1.94
0.0001
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Table 3: The hematological Profile for the studied groups
Groups
Normal
EAC
Parameter
Hb (g/dl)
14.5 ± 0.22
11.4 ± 0.19
*
p
p
Vehicle
BBIA-Co
Treated
13.1 ± 0.76
13.1 ±0.31
12.5 ± 0.36
--
0.001
--
NS
0.01
0.001
0.001
0.01
Ns
RBCs
(×106cell/mm3)
*
p
8.6 ± 0.09
6.7 ± 0.13
7.8 ± 0.47
97.6 ± 0.13
7.3 ± 0.2
--
0.001
NS
0.001
0.001
**
--
--
0.01
0.001
0.02
7.0 ± 0.4
23.8 ± 3.63
5.2 ± 0.53
7.1 ± 0.31
6.8 ± 0.16
--
0.001
0.02
NS
NS
--
0.001
0.001
0.001
**
p
WBCs
(×103cell/mm3)
*
p
**
p
*
p: p value with respect to normal group, **p: p value with respect to EAC group.
p value ≤ 0.05 is significant, NS: non-significant.
Biochemical Studies:
Liver and kidney Functions:
Results revealed significant change in almost all markers of liver function by EAC inoculation. Significant
increase in the mean values of ALT, AST and reduced total proteins, albumin and globulin was obtained after
15 days of EAC inoculation. Injection of BBIA-Co to EAC bearing mice had significantly adjusted mean values
of total proteins, albumin and globulin reaching mean values comparable to healthy animals (Table 4).
Concerning kidney function, no significant changes in the mean values of urea and creatinine were noted
between normal mice group and EAC bearing tumor, however, increased uric acid mean value was detected in
EAC bearing mice. Treatment by 40mg BBIA-Co/kg B.Wt had significantly restored the uric acid to normal
value (Table 4).
Table 4: Liver and kidney functions for the studied groups
Groups
Normal
EAC
Vehicle
Parameter
ALT (U/L)
29.4±1.83
50.4±2.27
42.8±0.98
*
p
0.001
0.001
**
p
0.02
AST (U/L)
57.7±3.22
102.6±10.89
68.6±1.59
*
p
0.001
0.01
**
p
0.001
ALP (U/L)
74.6±5.10
84.8±9.07
44.3±2.25
*
p
NS
0.001
**
p
0.001
Proteins (g/dl)
4.6±0.03
3.6±0.01
4.8±0.05
*
p
0.001
0.01
**
p
0.001
Albumin (g/dl)
2.1±0.07
1.7±0.01
2.3±0.02
*
p
0.001
0.02
**
p
0.001
Globulin (g/dl)
2.5±0.08
1.9±0.001
2.5±0.05
*
p
0.001
NS
**
p
0.001
Urea (mg/dl)
72.0±3.24
70.1±1.15
71.8±1.49
*
p
NS
NS
**
p
NS
Creatinine (mg/dl)
0.3±0.01
0.3±0.01
0.3±0.01
*
p
NS
NS
**
p
NS
Uric acid (mg/L)
2.7±0.10
5.9±2.71
3.6±0.36
*
p
0.01
NS
**
p
0.01
*
p: p value with respect to normal group, **p: p value with respect to EAC group.
p value ≤ 0.05 is significant, NS: non-significant.
BBIA-Co
Treated
54.1±4.60
0.001
NS
98.5±2.29
0.001
NS
76.2±4.60
NS
NS
4.2±0.04
0.001
0.001
1.9±0.03
NS
0.001
2.3±0.04
NS
0.001
63.3±2.61
0.03
0.02
0.3±0.01
NS
NS
3.2±0.12
0.01
0.001
58.2±5.06
0.001
NS
104.7±1.73
0.001
NS
86.2±2.97
0.01
NS
4.3±0.04
0.001
0.001
2.1±0.02
NS
0.001
2.1±0.04
0.01
0.001
59.0±1.68
0.001
0.001
0.3±0.001
0.001
0.001
2.6±0.05
NS
0.001
Serum glucose and lipid profile:
The current study demonstrated significant reduction in serum glucose in untreated EAC bearing mice
group. Treatment with BBIA-Co had resulted in elevated blood glucose to value greater than normal value.
Moreover, compared to normal mice group, significantly elevated triglycerides and VLDL mean values were
noted in all animal groups (Table 5).
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Table 5: Serum glucose and lipid profile for the studied groups
Groups
Normal
EAC
Vehicle
Parameter
Glucose (mg/dl)
116.2±4.33
85.2±3.07
98.2±0.63
*
p
0.001
0.01
**
p
0.01
Triglycerides (mg/dl)
61.4±3.94
123.6±10.96
95.8±6.09
*
p
0.001
0.001
**
p
NS
Cholesterol (mg/dl)
101.2±3.72
111.2±2.95
105.9±2.51
*
p
NS
NS
**
p
NS
VLDL (mg/dl)
12.2±0.82
28.4±2.83
20.8±1.14
*
p
0.001
0.001
**
p
NS
*
p: p value with respect to normal group, **p: p value with respect to EAC group.
p value ≤ 0.05 is significant, NS: non-significant.
BBIA-Co
Treated
145.3±4.92
0.001
0.001
119.3±5.86
0.001
NS
93.1±2.44
NS
0. 01
23.9±1.15
0.001
NS
190.0±4.23
0.001
0.001
131.0±7.68
0.001
NS
120.6±10.67
NS
NS
26.2±1.54
0.001
NS
Discussion:
Cancer is one of the leading causes of mortality worldwide. The failure of conventional chemotherapy to
affect major reduction in the mortality indicates that new approaches are critically needed. Currently the
curative effects of the exciting chemotherapy drugs are not good enough and they have plentiful side effects.
The major focus of research in chemotherapy for cancer includes the identification, characterization and
development of new and safe cancer chemo- preventive agents. In the current study, the antibacterial activity of
a novel synthetic cobalt complex of bromobenzaldehyde-iminacetophenone (BBIA-Co) was estimated. The in
vitro cytotoxicity of the complex against different cell lines was examined. Moreover, in vivo antitumor
activities of the complex against EAC inoculated mice; at a dose equivalent to 40 mg/kg body weight was
examined.
The present data indicated that the metal complex under investigation has the capacity to inhibit the growth
of the tested gram-positive (Staphylococcus aureus) and gram-negative (Escherchia coli) bacteria. The activity
of this Schiff base complex may arise from the presence of C=N and C=O moieties. The mode of action may
involve the formation of hydrogen bonding between these functional groups and the active centers of the cell
process [1].
The initial step of evaluating most anticancer agents is cell culture. The in vitro cytotoxicity of the metal
complex showed different antitumor activities against different human cancer cell lines. BBIA-Co exhibited
medium in vitro cytotoxicity, with IC50 values of equivalent to 25.0 µg/ml, 17.5 µg/ ml and 21.7 µg/ ml against
HPG2, A549 and PC3 cell lines, respectively.
To establish the validity of the metal aqua complex as anticancer agent, further studies were carried out to
demonstrate their in vivo cytotoxic activity against EAC cells inoculated in male albino mice. In this study, the
BBIA-Co complex had resulted in life span prolongation of EAC inoculated mice. The mean survival time
(MST) of untreated EAC bearing mice was 26.5 days. Because of rapid EAC cell division during the
proliferating phase, ascites fluid accumulated, resulting in host animal death due to the pressure exerted by the
tumor volume and/or the damage that resulted from the tumor itself [41,5]. Treatment with BBIA-Co complex
resulted in increased MST, reaching 47 days, with percentage increase in life span equivalent to ~77 %. This
result might point out to effectiveness of the complex as anticancer agent, since one of the reliable criterions for
judging the value of anticancer drug is the prolongation of animal's life span by more than 25% [21].
In our study, the increased food intake by mice bearing EAC tumor during the studied experimental period
probably may enhance the growth of transplanted EAC tumor cells, by enhancing glycolysis rate of the tumor,
Warburg’s effect [57,31,58]. It is well known that dietary energy restriction (DER) often referred to “as under
nutrition without malnutrition” is a potent inhibitor of the process of carcinogenesis. It has been hypothesized
that DER primarily targets neoplastic cells by reducing net energy status and particularly glucose metabolism,
therefore lower the metabolism of transformed cells, since more than 75% of tumor types relay on aerobic
glycolysis [57]. This finding was recently supported by Singh et al., [53]. The authors reported that dietary
supplementation of the glycolytic inhibitor, 2-diphosphoglycerate, to mice effectively inhibits tumorigenesis
same as DER effects.
Inoculation of EAC cells to mice, in the current research, resulted in significant increase in body
weight after 14 days of inoculation (33.4 %) compared to normal mice (14.4%), which could be attributed to
tumor burden. The Ehrlich tumor cells are aggressive rapidly growing cells [48] which can grow in different
mice strains [3]. The implantation of EAC cells induces local inflammatory reaction with increasing vascular
permeability resulting in edema, cellular migration and a progressive ascetic fluid formation [18]. The ascetic
fluid constitutes nutritional source for the cells, therefore, it is essential for tumor cell growth [50]. Treating
EAC group with BBIA-Co had prevented the accumulation of ascetic fluid resulting in reduction in weight gain
(17.8%); almost comparable to normal mice group; at the end of experiment.
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In cancer chemotherapy, the major problems that are being encountered are myelosuppression and anemia
[43,24]. In the current study, EAC bearing mice had significantly decreased RBCs count and hemoglobin
contents, indicating the existence of anemia. This may occur either due to iron deficiency or due to hemolytic or
myelopathic conditions [19]. After treating mice inoculated with EAC cells with 40 mg of BBIA-Co
complex/Kg, three times/week for two weeks, the changes in RBCs and Hemoglobin were reversed, indicating a
protective action on the hemopoietic system. Moreover, tumor induction resulted in significant elevation in
WBCs count while it was significantly decreased by treatment to values comparable to control mean value. The
effect of EAC cells inoculation on hematololgical profile in mice was reported by many authors [11,54]. The
reduction in WBCs count in animals bearing tumor after treatment was suggested to be one of the reliable
criteria for judging the efficiency of the drug as an anticancer agent [9].
The presence of tumors in experimental animals was found to affect many functions of the vital organs,
even when the site of tumor does not interfere directly with organ function (DeWys,1982). In the current study,
elevated serum ALT and AST activities were observed in EAC bearing mice. Elevated liver enzymes in EAC
bearing mice was previously reported by different researchers [22,12,11]. Our study indicated that treatment by
Co- complex did not ameliorate the activities of the liver enzymes. Moreover, injecting the complexes to healthy
animals had resulted in significant increase in ALT and AST activities, with AST mean values more than twice
that for ALT in all groups receiving BBIA- Co complex. ALT is found predominantly in the liver, with
clinically negligible quantities found in the kidneys, heart and skeletal muscles. On the other hand, AST is found
in the liver, cardiac muscle, kidneys and red blood cells. In this study as AST activity is higher than ALT
activity in the groups of animals receiving the complex under examination, this may reflect the existence of
muscle source of these enzymes, probably cardiac muscles, rather than liver inflammation. Furthermore, plasma
proteins yield most useful information in chronic liver disease, since the liver is the site of albumin synthesis
and also possibly of some α and β –globulins. In this study, EAC bearing mice exhibited decreased total proteins
mainly, albumin. This finding is consistent with other studies [30,2]. Treatment of tumor bearing mice with the
complexes under investigation had significantly restored the changes in total proteins and albumin toward
normal, suggesting restoration of liver function, which in term may support the suggestion of other sources for
elevated enzymes rather than the liver.
In the present work, no significant differences in serum urea or creatinine levels were noted, indicating
unchanged kidney function by EAC inoculation. On the other hand, remarkable increase in serum uric acid
mean value was obtained in EAC bearing mice group. Treatment with BBIA-Co had significantly restored uric
acid to normal values. Uric acid is the final product of purine metabolism and its circulating concentrations are
regulated by the balance in its production and excretion [56]. The elevated serum uric acid in EAC bearing
animals could be due to the malignant process itself, resulting from the increased nucleic acid turnover in the
rapidly proliferating diseased tissue [49].
The current study demonstrated that EAC inoculation to mice had resulted in a significant decrease in blood
glucose compared to normal mice. Most studies had observed lower serum glucose in tumor bearing mice
[40,10,26]. One reason for hypoglycemia could be an augmented consumption of glucose by the cells of the
tumor [13,39]. As indicated in the current study, treatment had resulted in increased serum glucose, although
levels highly exceeded those for normal control.
In the current study, EAC induced defects in lipid profile of mice, as manifested by elevated TAG and
VLDL. In this regards, several authors have presented evidences indicating that EAC had affected lipid
metabolism of the host animals. Highly significant increase in serum TAG, free fatty acids and ketone bodies in
tumor bearing mice were previously demonstrated [8,33,14]. Several mechanisms have been proposed by
different authors to explore the hypertriglyceridemia and elevated VLDL associated with EAC bearing mice.
Lyon et al., [34] attributed the elevated TAG to defective removal of VLDL-TAG. Others [29] reported that
fasting lowers plasma TAG in mice, but chronically lowered insulin levels and other hormonal changes that
followed reduced food intake might cause inhibition of VLDL-TAG removal from the circulation. The high
degree of lipemia in tumor bearing animals could be also due to mobilization of body fat for energy production
[42,36,37]. Another possible explanation for hypertriglyceridemia was the suggestion that the presence of tumor
leads to secretion of TAG in the form of VLDL; properly from the liver; which enters the peritoneal cavity.
TAG and cholesterol contained in VLDL are taken up and utilized by Ehrlich cells, suggesting that one function
of VLDL is to transport lipids from host tissue to the growing tumors [34]. In this study, although treatment
with BBIA-Co complex had ameliorated some of the abnormalities induced by EAC inoculation, however the
complex was unable to improve the changes in lipid profile.
The mode of action of the cobalt drug under investigation, [Co(BBIA) 2(H2O)4]2+, might be comparable to
that of the established cisplatin drug. Cisplatin, cis-diamminedichloroplatinum (II) [PtCl2(NH3)2], is a wellknown chemotherapeutic drug. Following administration, one of the chloride ligands is slowly displaced by
water, in a process termed aquation. The aqua ligand in the resulting [PtCl(H2O)(NH3)2]+ is itself easily
displaced, allowing the platinum atom to bind to bases. Of the bases on DNA, guanine is preferred. Subsequent
to formation of [PtCl(guanine-DNA)(NH3)2]+, cross-linking can occur via displacement of the other chloride
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Advances in Environmental Biology, 10(12) December 2016, Pages: 159-170
ligand, typically by another guanine. Its mode of action has been linked to its ability to cross-link with the
purine bases on the DNA; interfering with DNA repair mechanisms, causing DNA damage, and subsequently
inducing apoptosis in cancer cells. Cisplatin cross-links DNA in several different ways, interfering with cell
division by mitosis. The damaged DNA elicits DNA repair mechanisms, which in turn activate apoptosis when
repair proves impossible [18,15]. Also, various transition metal complexes were synthesized and checked for the
possibility to act as antitumor drugs. Our cobalt complex ([Co(BBIA) 2(H2O)4]2+) has proven to have promising
in vitro and in vivo cytotoxicity. Therefore, it is suggested that the cobalt complex, [Co(BBIA) 2(H2O)4]2+, could
presumably behave the same as cisplatin. Interestingly, the drug already has coordinated water molecules which
eliminate the need for the aquation step necessary for the cisplatin. Thus, the cobalt complex ion could easily
bind to the purine bases of DNA, causing DNA damage and subsequently inducing apoptosis in cancer cells.
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