Inhibitory Effect of Sulforaphane against Benzo(a)pyrene Induced Lung Cancer by Modulation of Biochemical Signatures in Female Swiss Albino Mice
D. Kalpana Deepa Priya,
Sulforaphane (SFN) is an organosulfur compound found in brassica vegetables. The present study was undertaken to investigate the defending role of sulforaphane at a dose of 9 μmoles/mouse/day against the cellular dysfunction in benzo(a)pyrene [B(a)P] (100 mg kg-1 b.w., i.p.) induced experimental lung carcinogenesis. The sub cellular derangements were assessed by the cytotoxic and cellular functional markers, lysosomal enzyme leakage and enzymes of nucleic acid metabolism. The concept of free radicals contributing to carcinogenesis was confirmed by evaluating thioredoxin reductase and heme oxygenase. The carcinogenic biomarkers were complemented by immunohistochemical analysis of cell proliferating nuclear antigen-Ki67 and measurement of serum carcinoembryonic antigen by ELISA. Our work proves the chemoprotective potential of sulforaphane in vivo against cellular derangements caused by B(a)P in the process of carcinogenesis.
to cite this article:
D. Kalpana Deepa Priya, R. Gayathri, G.R. Gunassekaran, S. Murugan and D. Sakthisekaran, 2011. Inhibitory Effect of Sulforaphane against Benzo(a)pyrene Induced Lung Cancer by Modulation of Biochemical Signatures in Female Swiss Albino Mice. Asian Journal of Biochemistry, 6: 395-405.
Received: February 11, 2011;
Accepted: May 16, 2011;
Published: July 29, 2011
Recently women have been diagnosed in increasing numbers than men of Lung cancer
which is often caused by exposure to cigarette smoke or other environmental
carcinogens. Of the many substances present in tobacco smoke, B(a)P is considered
a prototype polycyclic aromatic hydrocarbon (PAH), classic DNA damaging agent
and carcinogen. It is a ubiquitous pollutant found in amounts of 10 ng per cigarette
contributing about 200 ng/day for a pack-a-day smoker (Scherer
et al., 2000). The process of tumorigenesis is initiated when a replication-competent
cell acquires a mutation in a gatekeeping pathway that endows it
with a selective growth advantage. Many of the known gatekeepers were identified
through the study of unusual families with predispositions to specific types
of cancers (Knudson, 2002). It appears that the cells
of solid tumors must accumulate several rate-limiting mutations in cancer genes
to achieve malignant status. Each of these cells has multiple genetic abnormalities
and is capable of rapidly evolving variants to combat any therapeutic onslaught
(Nygren and Larsson, 2003).
Recent focus of cancer chemoprevention is on intermediate biomarkers capable
of detecting early changes that can be correlated with inhibition of carcinogenic
progression. Although Sulforaphane (SFN) is not a direct-acting antioxidant,
there is substantial evidence that SFN acts indirectly to increase the antioxidant
capacity of animal cells and their abilities to cope with oxidative stress (Dinkova-Kostova
et al., 2006). It is highly effective in preventing or reducing tumor
formation induced by carcinogens in animal models (Fahey
et al., 2002). Much of sulforaphanes cytoprotective enzyme
induction is thought to occur via the actions of the transcription factor Nrf2
(Fahey et al., 2002).
A potential strategy for diagnosing lung cancer, the leading cause of cancer-related death, is to identify metabolic signatures (biomarkers) of the disease. Such analysis will lead to new biological insights of the disease identifying potential therapeutic targets. With this initiative in mind the present study was undertaken to analyze the protection offered by sulforaphane against B(a)P induced biochemical alterations during the process of carcinogenesis in serum and tissue samples of cancer induced and treated female Swiss Albino Mice (SAM). This will further help us to analyze the metabolic state of the disease involving cellular and sub-cellular organelle dysfunction.
MATERIALS AND METHODS
Drugs and chemicals: Benzo(a)pyrene was purchased from Sigma Chemical Company, USA. Sulforaphane (>99% purity) was purchased from LKT Laboratories (St Paul, MN). All other chemicals used were of analytical grade obtained from Sisco Research Laboratories Pvt. Ltd., Mumbai, India and Glaxo Laboratories, CDH division, Mumbai, India.
Animals: Healthy female Swiss albino mice (4-6 weeks old) weighing 15-20 g were used throughout the study (2007-2010). Mice were acclimated to laboratory condition with regular temperature control ranging from 23±2°C and were given ad libitum access to balanced diet (Gold Mohor rat feed, Ms. Hindustan Lever Ltd., Mumbai) and water. All the experiments were performed in compliance with the regulation of our institutional Animal Care and Use Committee. They were maintained in a controlled environment condition of alternative 12 h light/dark cycles. This research work on female Swiss albino mice was sanctioned and approved by our Institutional animal ethical committee (IAEC/No-02/077/2007).
Treatment schedule: The animals were divided into five groups with six
mice in each group.
||Control animals treated with corn oil (vehicle) intraperitoneally
||Animals were treated with Benzo(a)pyrene [B(a)P] (100 mg kg-1
b.wt., dissolved in corn oil, intraperitoneally) thrice a week to induce
lung cancer. The method of cancer induction was adopted from Wattenberg
et al. (1997) and given intraperitoneally thrice a week at a
dose of 100 mg kg-1 b.wt. and treatment conditions were accordingly
decided in the current study
||Animals treated with sulforaphane (9 μmoles/mouse/day) on alternate
days for two weeks prior to first dose of the carcinogen and continued until
the 12th week and sacrificed (Pre-treatment group)
||B(a)P administered animals were treated with sulforaphane from the 12th
week to the 20th week (Post-treatment group)
||Animals treated with sulforaphane alone as in Group III served as drug
After the respective treatment periods the animals were sacrificed and their
blood, lung and liver tissues were used for the biochemical analyses. Both the
organs were excised immediately and washed in ice cold saline to remove any
extraneous matter and cleaned and blotted to dryness in Whatman No. 1 filter
paper. A 10% homogenate of lung and liver tissues were prepared in appropriate
buffers as necessitated by the protocols. Dilutions were decided based on the
protein concentration. A portion of the lung tissue was immediately stored for
immunohistochemical studies in 10% buffered formalin. The method of Lowry
et al. (1951) was adopted for the estimation of protein content in
the serum and tissue homogenates.
Enzymes of cellular integrity and cytotoxicity: The activity of alkaline
phosphatase was determined using disodium phenyl phosphate as the substrate
(King, 1965a) and was expressed as Fmoles of phenol liberated/min/mg
protein in tissue and IU L-1 in serum. AST and ALT were assayed by
the method of King (1965b) and expressed as μmoles
of pyruvate liberated/min/mg protein in tissue and IU L-1 in serum.
The assay of LDH was based on its ability to convert lactate to pyruvate in
the presence of the coenzyme nicotinamide adenine dinucleotide (NAD+)
(King, 1965c) and expressed as μmoles of pyruvate
liberated/min/mg protein in tissue and U L-1 in serum. Activity of
γGT was determined by the method of Orlowski and Meister
(1965) using L-γ-gutamyl-p-nitroanilide as the substrate and expressed
as nmoles of p-nitroaniline liberated /min/mg protein. Acid phosphatase was
assayed by the method of King (1965a) and the activity
was expressed as ìmoles of phenol liberated/min/mg protein under incubation
Lysosomal markers and nucleic acid enzymes: The activity of cathepsinD
was estimated by the method of Biber et al. (1981)
using haemoglobin as the substrate and expressed as μmoles of tyrosine
released/min/mg protein. βD-Glucuronidase activity was assayed by the method
of Kawai and Anno (1971) and expressed as μmoles
of p-nitrophenol liberated/min/mg protein.
5-Nucleotidase was assayed by the method of Luly et
al. (1992) and expressed as ìmoles of inorganic phosphorus liberated/min/mg
protein. ADA activity was measured by the spectrophotometric method described
by Guisti and Galanti (1974) and expressed as international
unit (IU L-1) in serum and ìmoles of NH3 liberated/mgprotein/hr
in tissue. XO was assayed by the method of Fried and Fried
(1966) and expressed as U mg-1 protein (1 unit corresponds to
the change of 0.1 OD/min by 1 mL of the enzyme).
Oxidative stress markers and carcinogenic markers: Thioredoxin reductase
activity was assessed by the methodology adopted by Holmgren
(1977) and expressed as U mg-1 protein (1unit corresponds to1
μmole of TNB formed/min). The activity of heme oxygenase was determined
by the method of Tenhunen et al. (1969) and expressed
as μmoles of bilirubin formed/min/mg protein based on its millimolar extinction
coefficient -27.7. The activity assay of aryl hydrocarbon hydroxylase was modified
from the method of Buening et al. (1981) and expressed
as pmoles of phenolic metabolite formed/min/mg protein in serum. Assay of Amylase
activity in serum and tissue was carried out by the method of Nelson
(1944) and expressed as ìmoles of maltose liberated/mg protein in
tissue and IU L-1 in serum.
Carcinoembryonic antigen (CEA): Quantitative estimation of CEA was based on the solid phase enzyme linked immunosorbent assay. CEA was assayed using UBI Magiwell enzyme immunoassay kit. The values were expressed as ng mL-1 serum.
Immunohistochemistry for Ki67: Immuno Histochemical analysis of Ki67 expression was carried out on paraffin sections of lung tissue using the B-SAP universal staining kit (Span diagnostic ltd, Surat, India). Initially, the sections were de-paraffinised in xylene and dehydrated in ethanol. After antigen retrieval the slides were incubated for 5 min with blocking solution (10% normal goat serum) at room temperature. Then the sections were incubated for 4 h with mouse monoclonal anti-Ki67 antibody (Sigma chemicals). Then washed with PBS and subsequently incubated with biotinylated secondary link antibody for 30 min at room temperature. Finally treated with Diaminobenzidine chromogen for 15 min and then washed with deionised water, counter stained with hematoxylin and mounted. Photographs were taken using Nikon microscope ECLIPSE E 400, model 115, Japan.
Statistical analysis: Data are presented as the Mean±Standard deviation (SD).One-way Analysis of Variance (ANOVA) was used to detect the significant changes between the groups. The student Least Significant Difference (LSD) method was used to compare the means of different groups and the significance was denoted by P value. A commercial software SPSS version 10.0 was employed to find out the statistical significance between the groups and p<0.05 was considered statistically significant.
Figure 1a represents the effect of sulforaphane on the activities of marker enzymes such as aspartate Aminotransaminase (AST), alanine Aminotransaminase (ALT), Acid Phosphatase (ACP) and Alkaline Phosphatase (ALP) in serum of control and experimental groups. A profound increase was noticed in the activities of all serum maker enzymes signifying p<0.001; p<0.01; p<0.01; p<0.001 for AST, ALT, ACP and ALP respectively in group II animals. On treatment with sulforaphane, a significant reduction (p<0.001) in the activities of the above mentioned enzymes were observed except for ACP signifying p<0.05 in group III and IV animals.
The activities of serum Aryl Hydrocarbon Hydoxylase (AHH) and adenosine deaminase were significantly increased (p<0.001) in the group II induced animals and decreased (p<0.01; p<0.05) respectively on treatment with sulforaphane as shown in Fig. 1b. In tissue a significance of p<0.001 for ADA was observed on treatment with sulforaphane in group III and IV animals.. Interestingly amylase was found to be decreased (p<0.001) in the serum but increased (p<0.001) in tissue of cancer induced animals and increased (p<0.01 and p<0.05) in serum in group III and IV animals treated with sulforaphane. A reverse effect was seen (p<0.001) in tissue of pre-and post-treated animals. Drug control animals showed no significance when compared with control animals.
Figure 1c illustrates the alteration in the activity of carcinogenic marker (γGT) in serum, lung, liver and 5nucleotidase activity in liver of experimental animals. In group II animals γGT was profoundly increased signifying with p<0.001 in serum and lung and with p<0.01 in liver and similar observation was made with 5nucleotidase in liver of group II animals. Pre-treatment with sulforaphane in group III animals decreased the activity of γGT signifying (p<0.001) in the serum and lung and (p<0.01) in liver. Group IV animals showed a similar decline (p<0.05) for γGT in serum and both tissues. A considerable reduction with p<0.05 and p<0.01 for 5 nucleotidase was observed in the group III and IV animals groups respectively. The sulforaphane alone treated animals (group V) did not show any significant changes when compared with control animals (group I).
||Effect of sulforaphane on the (a) serum and (b) and (c) carcinogenic
biomarkers of control and experimental mice. Values are expressed as Mean±SD
(n = 6); a: as compared with group I; b and c: as compared with group II;
*p<0.05; #p<0.01; $p<0.001; NS = Not significant
Figure 2a describes the modulations in the activities of liver function assessment enzymes. These include AST, ALT and ALP. In group II animals, the increased activities of ALT and AST (p<0.01) along with ALP (p<0.001), were found to be decreased significantly (p<0.05) for ALT and (p<0.001) for AST and ALP in group III and IV animals as compared to group I animals. Group V animals did not show any significant change when compared with control animals (group I).
Figure 2b illustrates the alteration in the activities of cytotoxic markers-LDH and ACP in lung and liver. LDH was profoundly increased signifying with p<0.001 in both tissues and acid phosphatase was increased with p<0.001 in lung and p<0.05 in liver in group II cancer induced animals. Their decrease was significant in the pre-treated and post-treated group signifying p<0.001 for LDH in both tissues. A reduction signifying p<0.001 on pre-treatment and p<0.01 on post-treatment for ACP in lung and p<0.05 in liver of group III and IV sulforaphane treated animals was observed was observed. The drug control group showed no significant changes.
||Effect of sulforaphane on the tissue (a) functional and (b)
cytotoxic markers of control and experimental mice. Values are expressed
as Mean±SD (n = 6); a: as compared with group I; b and c: as compared
with group II; *p<0.05; #p<0.01; $p<0.001;
NS = Not significant
Figure 3a explains the effect of sulforaphane on the lysosomal markers (Cathepsin D and βD-glucuronidase) and nucleic acid enzymes, 5Nucleotidase(5N) and Xanthine Oxidase (XO) in Lung tissue of control and experimental animals. A significant increase in βD-glucuronidase (p<0.001) and cathepsin D (p< 0.05) was observed in group II carcinogen administered animals when compared with controls. Likewise a radical decrease (p<0.01) for βD-glucuronidase was found in group III and IV animals. A noticeable increase in the group III (p<0.05) and group IV (p<0.001) for cathepsin D was noticed. A significant increase in the activities of 5N and XO (p<0.001) were observed in group II induced animals. On pre-treatment with sulforaphane the activities decreased (p<0.001). There was a slight difference in the alleviation in the group IV animals for these enzymes representing a significance of p<0.05 for 5N and p<0.01 for XO. Drug control animals showed no significance when compared with control animals.
Figure 3b depicts the modulation of oxidative stress markers-Thioredoxin Reductase (TRxR) and Haem Oxygenase (HO) associated with carcinogenesis. The activities of these enzymes were found opposing in the tumor induced animals where a significant decline was noticed with TRxR (p<0.01) and increase with HO (p<0.001).A similar opposing effect in the reverse order was seen on treatment with sulforaphane in the pre-(p<0.05) and post-treated(p<0.01) group for both enzymes.
Figure 3c shows the effect of sulforaphane on the expression of the fetal antigen Carcino Embryonic Antigen (CEA) in the serum of control and experimental mice. A significant increase in the expression of CEA in serum of cancer bearing group II animals when compared with control group I animals was detected. On treatment with sulforaphane this was decreased in group III and group IV animals when compared with group II animals. No significant changes were noticed in group V animals when compared with group I control animals.
Figure 4 represents the microphotographs of lung sections of the different experimental groups showing immunoreactivity to the cell proliferating nuclear antigen-Ki67. Group II slide has 4+ degree of positive reactivity when compared to the 2+ score in the post-treated group. The pre-treatment group III and drug control group IV showed no reactivity as seen in group I control slide.
||Effect of sulforaphane on (a) lysosomal enzymes, nucleic acid
enzymes, (b) oxidative stress markers and (c) serum CEA levels of control
and experimental animals. Values are expressed as Mean±SD (n = 6);
a: as compared with group I; b and c: as compared with group II; *p<0.05;
#p<0.01; $p<0.001; NS = Not significant
||Immunoreactivity to Ki 67 in control and experimental animals.
(a) Group I: Control animals showing absence of immunoreactivity for Ki67
(200X), (b) Group II: B(a)P treated animals showing a high gradation of
4+ positivity confined to the nucleus (200X) and (c) Group IV: Sulforaphane
post-treated animals with highly reduced immunoreactivity graded 2+ positivity
for Ki67 (200X)
The present study reflects the biochemical response of a transformed tissue
involving various cellular derangements by experimental induction of lung carcinogenesis
in mice. The aspartate and alanine aminotransferases (AST and ALT) are important
enzymes of liver whose activities are related with the amino acid homeostasis.
Their elevation in serum has been attributed to the damaged structural integrity
of hepatic cell membrane as reported earlier (Gupta et
al., 2006). (Chidambara Murthy et al., 2005).
In this study, we found that exposure to B(a)P brings about the hepatocellular
damage due to oxidative stress leading to increased enzyme activity which is
evident from a progressive increase in the serum levels of ALP, AST and ALT.
AST being a key mitochondrial enzyme, its increase in the serum levels would
indicate excessive mitochondrial proliferation (Hassanein,
2004). Treatment with SFN in group III and IV significantly reduced the
elevated levels of the enzymes towards their respective normal values indicating
rehabilitation of plasma membrane and as well as repair of hepatic tissue damage
induced by B(a)P.
Establishing the activities of some DNA turnover enzymes and free radical metabolizing
enzymes in cancerous tissues seems to be of particular importance to elucidate
possible relations between cancer and free radical metabolisms. In several studies,
ADA was found increased in cancerous tissue and cells compared to non-cancerous
ones (Camici et al., 1990) and this coincides
well with present results which reflects an accelerated purine turnover and
high salvage pathway activity of nucleic acid metabolism associated with tissue
hyper proliferation in B(a)P induced pulmonary pathogenesis. Also a high XO
activity recorded in group II B(a)P induced animals is due to increased conversion
of Xanthine dehydrogenase to oxidase (Sushma et al.,
2006) . This may be an attempt to lower salvage pathway activity which is
vital for rapid DNA synthesis in cancer cells. The treatment with SFN had therapeutic
consequences in B(a)P induced tumorigenesis with a concordant decrease of the
enzymes, reflecting a decreased purine turnover and hence the proliferation
rate in group III and IV animals.
A significant increased activity of amylase in the tissue (Lung) might be due
to its increased local production. Likely an increase in the serum was not observed
in the cancer induced group. This might be due to the enzyme modification caused
by the toxic metabolites formed during B(a)P activation leading to its functional
impairment. The marker enzymes such as AHH, ADA, GGT, 5-ND and LDH are
specific indicators of lung damage (Tessitore et al.,
1994).The increase in their activities may be due to the increased tumor
incidence (Ravichandran and Ramanibai, 2008). Also damage
to plasma membrane causes the leakage of these enzymes from the cytosol to the
blood stream (Shanmugarajan et al., 2008). Lactate
dehydrogenase is recognized as a potential tumor marker in assessing the progression
of the proliferating malignant cells due to its increased turnover. Gamma glutamyl
transpeptidase (γ-GT) is not only useful in diagnosis but also has prognostic
value in malignancies such as lung cancer and malignant melanoma. Its increase
in group II animals was to compensate the decreased GSH levels and an inhibitory
effect was posed by SFN in group III and IV animals which could be due to its
stimulatory effect on the GSH biosynthesis, hence reduced the depletion of GSH
from the cells by a autocrine positive feed back loop mechanism.
Intracellular Acid Phosphatase (ACP) and βD-glucuronidase (βGR) are
largely confined to lysosomes. Their activities correlate and primarily respond
to cellular injury and inflammation (Kumar et al.,
2005). Their increase in serum indicate an enhanced Golgi activity and peroxidation
in lysosomal membranes after exposure to B(a)P causing membrane lysis leading
to enzyme leakage. Cathepsin D, an endopeptidase was found recently to be an
important regulator of apoptosis acting as a direct activator of caspase 3 and
9 (Minarowska et al., 2007). In the current observation,
its mild release was noticed in the cancer induced group II animals due to increased
lipid peroxidation by B(a)P. This response was further boosted upon treatment
with SFN in group III and IV indicating the selectivity of the phytochemical
in initiating apoptosis and further support the emerging picture of Cathepsin
D as an important mediator of programmed cell death.
Thioredoxin reductases (TrxRs) play an important role in multiple cellular
events such as ROS detoxification, oxidoreductase activities and cytokine effects
(Yodoi, 2000) related to carcinogenesis including cell
proliferation, apoptosis and cell signaling. HO-1 displays antioxidant, anti-apoptotic
and anti-inflammatory effects and appears to have a complex role in angiogenesis
(Prawan et al., 2005). In present study both
enzymes were slightly elevated as a means of compensatory adaptation to the
depleted antioxidant enzymes and especially GSH (Priya et
al., 2011) in B(a)P treated mice. Increased levels of TrxR have been
reported in many different tumors with a correlation to malignancy and poor
prognosis (Kahlos et al., 2001). On treatment
with sulforaphane, TrxR activity was still increased due to the formation of
more SFN-GSH conjugate as reported earlier by Zhang and
Callaway (2002). This acts as a driving force for the formation of reduced
glutathione. The antioxidant action of hemeoxygenase is favored by the breakdown
of heme proteins thus reducing the lipid peroxidation and the production of
ROS in SFN treated animals. Carcinoembryonic antigen is a cell surface glycoprotein
expressed in fetal tissues and is transcriptionally silent in adults and its
elevated levels suggest that CEA could play an important role in cancer progression
(Jessup et al., 1999). The observed significantly
high levels of CEA in cancer bearing animals indicated the progression of tumor
growth. A decrease in CEA level, upon treatment is associated with a better
survival as seen in group III and IV animals. Ki-67 protein is an excellent
marker for determining proliferating cells in human and animal neoplasm (Ozaki
et al., 2007). In the present study, the Ki-67 labeling index increased
in group II animals indicating cell proliferation as it is expressed in all
phases of cell cycle except in the resting G0 and early G1 phases. It has been
previously reported to be strongly associated with the percentage of growth
fraction (Petrowsky et al., 2001) and poor prognosis
in pulmonary adenocarcinomas. The confirmation of adenocarcinoma by B(a)P was
complemented by CEA levels and degree of Ki67 positivity.
In summary, we were able to analyze different biochemical signatures in serum and tissues of mouse model of lung cancer using different biochemical methodologies. The biomarkers analyzed in the present study have their metabolic origins in various biochemical pathways which are important for the assessment of the risk involved in the process of carcinogenesis. The importance of this study is that it establishes the feasibility of using such biomarkers to detect the initiation of lung carcinogenesis which involves various cellular dysfunctions in the target and non-target tissues.
One of the authors Ms. D. Kalpana Deepa Priya, wishes to gratefully acknowledge the support given by the University Grants Commission, New Delhi, in the form of UGC scholarship under the UGC XI Plan Scheme of Research Fellowship in Sciences for Meritorious Students.
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