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Journal of Herbmed Pharmacology
J Herbmed Pharmacol. 2018; 7(4): 225-235.
Anticancer potential of Tinospora cordifolia and
arabinogalactan against benzo(a)pyrene induced pulmonary
tumorigenesis: a study in relevance to various biomarkers
Vandana Mohan
ID
, Ashwani Koul*
ID
Department of Biophysics, Basic Medical Science Block ΙΙ, South campus, Panjab University, Chandigarh, India
*Corresponding author: Prof. Ashwani Koul, Phone: +91 0172
2534124, Email: drashwanikoul@yahoo.co.in, ashwanik@pu.ac.in
Implication for health policy/practice/research/medical education:
Tinospora cordifolia plant and its various bioactive components have wide range of pharmacological properties. Aqueous extract
of Tinospora cordifolia stem and arabinogalactan (AG), its bioactive polysaccharide, act as anti-cancer agents and have the
potential for preparation of new drugs against lung cancer.
Please cite this paper as: Mohan V, Koul A. Anticancer potential of Tinospora cordifolia and arabinogalactan against benzo(a)
pyrene induced pulmonary tumorigenesis: a study in relevance to various biomarkers. J Herbmed Pharmacol. 2018;7(4):225-235.
doi: 10.15171/jhp.2018 .35.
Introduction: Aqueous Tinospora cordifolia stem extract (Aq.Tc) and arabinogalactan (AG),
its bioactive polysaccharide, which are antioxidant remedies were evaluated on pulmonary
cancer and associated tumor markers.
Methods: Mice were randomly segregated into 6 groups. Group I: animals served as control.
Group II: animals which were administered Aq.Tc extract (200 mg/kg, orally), thrice a week.
Group III: animals which received AG (7.5 mg/kg, orally) thrice a week. Group IV: animals
which were instilled with benzo(a)pyrene (B(a)P) (50 mg/kg, orally) twice within an interval
of 2 weeks. Group V: animals which received Aq.Tc extract as in group II, along with B(a)P
after 2 weeks of Aq.Tc administration. Group VI: animals which received AG as in group III
along with B(a)P after 2 weeks of AG administration.
Results: As expected, B(a)P treated mice exhibited high tumor incidence and multiplicity
with concomitant increase in serum/plasma markers like carcinoembryonic antigen (CEA),
circulating tumor DNA (ctDNA), lactate dehydrogenase (LDH) and tumor necrosis factor.
However, Aq.Tc and AG supplementation to B(a)P abused animals significantly attenuated
these parameters at different stages of cancer, depicting their anti-cancer effects in lung
carcinogenesis. Also, treatment of Aq.Tc and AG to tumor bearing mice reduced the degree
of histopathological alterations as compared to B(a)P installed mice. The apoptotic index
in case of Aq.Tc and AG fed mice treated with B(a)P was higher as compared to only B(a)P
treated mice. Further it was obser ved that Aq.Tc could induce higher deg ree of apoptosis when
compared to AG group, suggesting Aq.Tc as a more effective modulator of tumorigenesis.
Conclusion: Overall, these findings substantiate the chemopreventive potential of Aq.Tc and
AG against lung tumorigenesis. Aq.Tc was found to be more effective than AG in modulating
the process of lung carcinogenesis as ref lected by various observations.
A R T I C L E I N F O
Keywords:
Arabinogalactan
Benzo(a)pyrene
Lactate dehydrogenase
Medicinal plant
Tinospora cordifolia
Article History:
Received: 10 January 2018
Accepted: 20 June 2018
Article Type:
Original Article
A B S T R A C T
Introduction
Lung cancer is the prime cause of cancer mortality and
is a growing economic burden worldwide (1). Primary
factors that lead to induction of lung cancer include use of
tobacco, automobile exhausts, occupational exposure and
indoor air pollution (2). Polycyclic aromatic hydrocarbons
(PAHs) are the major constituents of these pollutants
(3). One of the most potent PAH, Benzo(a)pyrene {B(a)
P} is known to induce lung cancer in humans as well as
experimental animals (4).
In spite of various advancements in lung cancer
diagnosis and treatment, survival rates remain low (5).
Generally, tumor size and its extent of invasion detected
histopathologically (biopsy) is considered to be the gold
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Mohan and Koul
standard for evaluating the disease (6). Since the procedure
of biopsy is invasive and could add to the agony of the
patient, different serological markers of tumor could be
taken into account for specific diagnosis and evaluation
of progress of cancer (7). Sensitive and specific diagnostic
markers of lung cancer could facilitate the early detection
of the cancer lowering the associated mortality rates (8).
Various serum markers include circulating tumor DNA
(ctDNA), carcinoembryonic antigen (CEA), neuron
specific enolase (NSE), carbohydrate antigen 125 (CA125)
and squamous cell carcinoma antigen (SCC) (9). These
markers when combined with other diagnostic tools
could classify tumor into different molecular subtypes and
monitor disease relapse and treatment response (10).
Since, there is no magic bullet to combat cancer, lung
cancer is no exception to it, never the less, the risk
could be reduced by eliminating contact or exposure
to putative carcinogens. But this may not be feasible
always, considering the quality of air, soil and water in
the present scenario. Therefore, it presses the need for
adopting other practical measures to control the dreadful
disease. The complexity of this hyperproliferative disease
necessitates the adoption of secondary prevention as a
rational way to control it (11). The use of botanicals in the
form of vegetables, fruits and other dietary supplements
as chemopreventive agents seem to be relevant (12).
Chemoprevention is a reasonable and cost-effective
approach to decrease cancer mortality by inhibiting the
pre-cancerous events and/or delaying its progression (13).
Most of the drugs that are available to combat cancer have
shown severe side effects. Synthetic chemopreventive
drugs are cytotoxic, immune suppressants and may cause
a variety of ill effects in various organs of the body (14).
Various drugs that are currently being used are based
on folk remedies and subsequent ethnopharmacological
studies. There are more than 100 drugs of known
structure that are extracted from medicinal plants. As
modern medicine came up, people started looking for
active components from the crude extract but due to
their higher/acute side effects they are not much being
used (15). Various medicinal plants have been identified
for their anticancer properties and in varied number of
ailments (16,17). Tinospora cordifolia is a known medicinal
plant having a wide spectrum of pharmacological activity.
It is commonly known as ‘Guduchi’ or ‘Amrita’ and is
considered as an important drug of Indian Systems of
Medicine (18). Different parts of this plant including
leaves, roots and stem have been widely used for their
variety of medicinal properties (19). However, use of
stem has been widely reported, so far (20). T. cordifolia
is known to exhibit anti-inflammatory and anti-cancer
properties in various experimental models (21). There
are several experimental reports which document the
anticancer potential of aqueous, methanolic as well as
ethanolic extracts of T. cordifolia in various cancer models
(22,23). One of the active constituents in its polysaccharide
fraction is arabinogalactan (AG) (24). Its potent biological
activity and immune-enhancing properties have received
increased attention as a clinically useful nutraceutical
agent (25). AG has several properties which make it an
ideal adjunct supplement to be considered in cancer
protocols (26).
Aqueous extract of T. cordifolia is enriched in the
polysaccharide fraction containing AG when compared
with other types of extract (27). Many biological activities
of T. cordifolia have been attributed to its polysaccharide
fraction (28). The above mentioned observations warrant
further investigations with respect to preparations rich in
AG or its use as such active principle.
The polysaccharide fraction of T. cordifolia in lung has
shown profound anti-metastatic activity against lung
cancer cell lines (29). However, not much has been
explored about its effect in in vivo lung cancer models and
the underlying mechanism involved. Considering this, the
present study was designed to assess the effects of aqueous
T. cordifolia extract and its bioactive polysaccharide AG in
in vivo b enzo(a)pyrene induced pulmonary carcinogenesis
model.
Material and Methods
Chemicals and kits
1,1-diphenyl 2-picrylhydrazyl (DPPH), 2,2’-azino-bis(3-
ethylbenzothiazoline-6-sulphonic acid) (ABTS), Benzo(a)
pyrene (B(a)P) and AG (99% pure) were obtained from
Sigma-Aldrich Co. (St. Louis, MO, USA). Rest of the
chemicals used were of analytical grade. Terminal
deoxynucleotidyl transferase dUTP nick end labelling
(TUNEL) assay kit was procured from Trevigen Inc.
(Gaithersburg, Maryland, USA). FitAmpTM Circulating
DNA Quantification kit was procured from Epigenetek
(Farmingdale, New York, USA).
Preparation of aqueous Tinospora cordifolia stem extract
Tinospora cordifolia stems were collected from the Panjab
University Botanical Garden after identification by a
qualified Botanist. Aqueous powder of T. cordifolia was
prepared according to method described by Tiwari et al,
with minor modifications (30,31). Dried Aq. Tc powder
(500 mg) was reconstituted in 10 mL of distilled water and
was subjected to preliminary screening for the presence of
phytoconstituents by standard protocol (32).
Free radical scavenging activity assays
Free radical scavenging activity of Aq.Tc as well as AG was
analysed using DPPH (1,1-diphenyl 2-picrylhydrazyl)
radical scavenging assay (33) and ABTS radical cation
(ABTS·+) assay (34).
Animals and experimental conditions
Male Balb/c mice (25–30 g) were procured from the Central
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Anticancer eects of Tinospora cordifolia in lung cancer
Animal House, Panjab University, Chandigarh (Approval
no PU/IAEC/S/15/61 Panjab University, Chandigarh).
Mice were housed in polypropylene cages bedded with
sterilized rice husk and were provided with standard pellet
diet (Ashirwad Industries Ltd., Ropar, Punjab, India) and
water ad libitum with standard conditions of relative
humidity (50%–60%) and temperature (24±2°C). Mice
were acclimatized to the experimental conditions for one
week after which various treatments were given.
Experimental design
Mice were randomly segregated into 6 groups (n=6-
8). Group I animals served as control and received no
special treatment. Group II animals were administered
aqueous T. cordifolia extract (Aq.Tc) (200 mg/kg, orally)
on the alternate days (thrice a week). Group III animals
were administered AG (7.5 mg/kg, orally) on alternate
days (thrice a week). Group IV animals were instilled
with B(a)P (50 mg/kg, i.p.) twice, within an interval of
two weeks. Group V animals received Aq.Tc extract as
in group II, along with B(a)P was installed after 2 weeks
of Aq.Tc administration following the same protocol as
for group IV. Group VI animals received AG as in group
III along with B(a)P was instilled after 2 weeks of AG
administration following the same protocol as in group
IV. Changes in body weight, diet and water intake were
observed every week in all the groups throughout the
treatment period. Dose of Aq.Tc in the present study was
standardized according to the available literature (200 mg/
kg) (35). Dose of AG was deduced considering the amount
of AG (active polysaccharide) present in 200 mg of Aq.Tc
which amounted to 7.5 mg/kg (Figure 1A).
Tumor assessment studies
Tumor statistics
At the end of the treatment period (22 weeks), the lung
tissue/lung tumors were excised and analyzed for (a)
tumor incidence (b) tumor size (c) tumor multiplicity
following the protocol of Gupta et al (36).
Histopathological evaluation
Lung tissue/tumors were excised at the end of 10th and
22nd weeks, fixed in 10% formaldehyde in phosphate
buffer saline (pH 7.4) for Haematoxylin and eosin (H&E)
staining as per the conventional laboratory procedures
(37). H&E stained slides were evaluated under light
Figure 1. (A) Animal treatment schedule to study the effect of Aq.Tc and AG in B(a)P induced pulmonary carcinogenesis. (B) Alterations
in average body weight of the animals during the treatment period. Data were represented as Mean ± S.D. (n=6-10)
(A) (A)
(B)
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228
Mohan and Koul
microscope (LEICA DM 3000).
Tumor markers
Various tumor markers were estimated in the plasma/
serum of mice from different treatment groups. Blood
samples from the mice of various treatment groups were
collected by retro-orbital puncture. Prior to retro-orbital
puncture, a drop of 0.5% pro-paracaine hydrochloride
ophthalmic solution (Sunways Pvt. Ltd. India) was applied
to anesthetize the eyes so that pain was reduced.
Carcinoembryonic antigen
Quantification of tumor marker CEA was estimated
in blood serum using the Qayee-Bio Mouse
carcinoembryonic antigen ELISA kit. The detection range
of the kit lies between 78 pg/mL to 5000 pg/mL.
Circulating tumor DNA
Circulating tumor DNA was quantified using the
FitAmp™ Circulating DNA Quantification kit for mouse.
The linear detection range of the kit lies between 0.1 ng
to 100 ng (1-1000 ng/mL) in 96-well plate assay. DNA
was fluorescently quantified at Ex 480-500 and Em 520-
550 nm using a fluorescence microplate reader (Biotek
Synergy H1 microplate reader).
Lactate dehydrogenase
The specific activity of Lactate dehydrogenase (LDH)
in blood serum was estimated according to the method
described by Bergmeyer and Bernt (38) using molar
extinction coefficient 6.22 mM-1cm-1.
Quantification of tumor necrosis factor-alpha
Quantitative estimation of tumor necrosis factor-α
(TNF-α) in blood serum samples of various treatment
groups was carried out by using in vitro enzyme linked
immunosorbent assay (ELISA) (Ray Biotech). The
minimum detectable dose of mouse TNF-α is 30 pg/ml.
TUNEL assay
The TUNEL assay was performed on deparaffinised and
rehydrated lung sections with the TACS-XL DAB in situ
apoptosis detection kit according to the manufacturer’s
specifications. Apoptotic index was calculated as the
percentage of TUNEL (+ve) cells were quantified over the
total number of cells in the selected lung tissue section.
Statistical analysis
The data were represented as mean ± standard deviation
(SD). and analysed by one-way ANOWA followed by LSD
post hoc test using the SPSS software package (version 16)
for windows (SPSS Inc., Chicago, IL).
Results
Phytochemical screening
In the present study, preliminary phytochemical analysis
Figure 2. (A) Radical scavenging effects of Aq.Tc. Extract and AG
on ABTS radicals. (B) Radical scavenging effects of Aq.Tc. extract
and AG on DPPH radicals. Data were represented as mean ± SD
(n = 3).
of Aq.Tc stem extract showed the presence of alkaloids,
phenols, flavonoids, carbohydrates, and proteins.
However, steroids were found to be absent in the extract.
Free radical scavenging activity
In DPPH and ABTS free radical scavenging assay Aq.Tc
extract exhibited free radical scavenging activity in a
concentration dependent manner when compared with
other strong antioxidants like BHT and tannic acid
(Figure 2A and 2B). It showed a higher antiradical activity
with IC50 of 432 µg and 49.8 µg in DPPH and ABTS assays,
respectively. However, AG showed a greater antiradical
activity at higher concentrations when compared with
Aq.Tc, BHT and tannic acid. The IC50 of AG was found
to be 648 µg and 384.17 µg in DPPH and ABTS assay,
respectively (Ta b l e 1).
Body status, diet and water intake
Mice in all the experimental groups did not show any
significant change in the diet and water consumption
throughout the treatment period. A considerable decrease
in the body weight of animals in B(a)P group was observed
when compared with their control counterparts. There
was no significant difference in the body growth patterns
in animals treated with Aq.Tc and AG i.e., Aq.Tc, AG, B(a)
P + Aq.Tc and B(a)P + AG group when compared with
control group (Figure 1B).
(A)
0
20
40
60
80
100
120
10 20 50 100 200
% inhibition activity
Conc (µg/uL
Aq.Tc extract AG BHT Tannic acid
0
10
20
30
40
50
60
70
80
90
100
10 20 50 100 200
% scavenging activity
Conc (µg/µL)
Aq.Tc extract AG BHT Tannic acid
(B)
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Anticancer eects of Tinospora cordifolia in lung cancer
Morphological changes in lung/lung tumors
Normal architecture was observed in Aq.Tc and AG treated
lung when compared with control animals (Figure 3A).
However, in B(a)P challenged mice lung, large number
of superficial lesions and hematomas were observed.
Prominent tumor nodules around different lobes of lung
were also present (Figure 3B). Mice bearing tumors co-
administered with Aq.Tc and AG showed a significant
reduction in tumor nodules and tumor mass (Figure 3C,
D, E and F, respectively).
Tumor statistics
After 22 weeks of the B(a)P treatment, the mice from B(a)
P, B(a)P +Aq.Tc and B(a)P + AG group developed lung
tumors, whereas no tumor/lesion was observed in control,
Aq.Tc and AG group.
The total number of tumors/lesions formed in various
experimental groups and the associated parameters are
summarised in Table 2.
Histopathology
Tenth week of B(a)P treatment showed loss of normal
pulmonary architecture and higher number of hyper
chromatic nuclei with air space enlargement (Figure 4B).
However, in animals from B(a)P +Aq.Tc and B(a)P+AG
groups showed decreased number of hyperchromatic
irregular cells in the alveolar wall at 10th week of
treatment (Figure 4D-F). Histopathological changes in
the pulmonary tissue at 22nd week post instillation of B(a)
P revealed extensive proliferation of alveolar epithelium,
large number of eosinophilic macrophages infiltrating
the pulmonary parenchyma with more number of hyper
chromatic nuclei in the alveolar wall (Figure 5B). Number
of hyperproliferative cells in B(a)P + Aq.Tc and B(a)P +
AG group decreased when compared to B(a)P group at
Table 1. IC50 values of Aq.Tc. extract, AG, BHT and tannic acid using
ABTS and DPPH scavenging activity
Extract/
Compound
IC50 value (µg/µL)
ABTS assay
IC50 values (µg/µL)
DPPH assay
Tannic acid
BHT
Aq.Tc
AG
14.24
49.80
180.66
384.17
103.21
282.40
432.52
648.50
Figure 3. Gross morphology of lungs in mice of different treatment
groups.
(A) Control, (B) Pulmonary lesions in B(a)P treated mice, (C),
Aq.Tc, (D) B(a)P treated mice co-administered with Aq.Tc. extract,
(E) AG, (F) B(a)P treated mice co-administered with AG. (arrow
indicates tumor).
A
C
EF
D
B
Table 2. Eect of Aq.Tc and AG on B(a)P induced lung tumorigenesis at end of the treatment period where (n=6-10)
Parameters/Groups Control Aq.Tc AG B(a)P B(a)P+Aq.Tc B(a)P+AG
Tumor incidence (%) - - - 100% 66.6% 83.3%
Tumor mulplicity - - - 2.45±0.365 1.22±0.057 * 1.52±0.125 **
Total no of tumors - - - 96 53 71
Big tumors (>2 mm) - - - 83 44 63
Small tumors (<2 mm) - - - 13 9 8
Data is expressed as Mean±SD and are analysed by Student’s t test.
* P ≤ 0.01, **P < 0.05 with regard to B(a)P.
22nd week of treatment (Figure 5D-F).
Tumor markers
Carcinoembryonic antigen
Instillation of B(a)P showed a significant (P ≤ 0.001)
increase in the CEA levels in a time dependent manner
when compared with the rest of the groups (158±1.36
to 288± 21.0 pg/mL). However, in B(a)P+AG group the
increase was less intense (162 ± 6.20 to 228 ± 2.12 pg/mL).
Interestingly, the levels of CEA in B(a)P +Aq.Tc group
dropped from 165 ± 6.20 to 143±16.4 pg/mL (Table 3).
Circulating tumor DNA
A significant increase in ctDNA levels was observed in
B(a)P group at different time intervals when compared
with other respective groups (1.15 ± 0.012 to 3.28 ± 0.140
ng/mL). However, Aq.Tc administration to the cancer
bearing animals, significantly (P ≤ 0.001) lowered the
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230
Mohan and Koul
levels of ctDNA to their near normal values. Also, the
levels of ctDNA were found to significantly (P ≤ 0.001)
decreased in B(a)P + AG animals when compared to the
B(a)P group (Tab l e 3).
Lactate dehydrogenase
A significant increase in LDH activity in B(a)P group was
observed when compared to various treatment groups
(259 ± 54.1 to 628 ± 16.5 IU/L). However, treatment of
Aq.Tc to the cancer bearing animals significantly (P ≤
0.001) restored the levels of LDH to their near normal
values. Also, the levels of LDH were found to significantly
(P ≤ 0.001) reduced in B(a)P+AG animals when compared
to the B(a)P group (Tab l e 3).
Quantification of TNF-alpha
TNF-alpha levels were significantly increased in B(a)
P group when compared to control, Tc and AG groups
at different time intervals. Whereas, Aq.Tc treatment to
cancer bearing animals significantly (P ≤ 0.001) restored
the level of TNF-alpha to their near normal values. Also,
the levels of TNF-alpha significantly decreased (P ≤ 0.001)
Figure 4. Hematoxylin and eosin stained lung tissue at 10th week
of treatment at 200 X.
(A). Control mice showing normal histoarchitecture, (B) B(a)P
treated mice, (C) Aq.Tc treated mice, (D) B(a)P installed mice
co-treated with Aq.Tc, (E) AG treated mice, (F) B(a)P installed
mice co-treated with AG. SB; small bronchiole, SA; single
squamous epithelium, AW; alveolar wall, AS; alveolar space, AD;
Widespread alveolar destruction, N; thinning of alveolar walls,
T; thickening of alveolar wall, IAS; increased alveolar sac, TB;
thickened bronchiolar epithelium.
Figure 5. Haematoxylin and Eosin stained lung tissue/lung
tumors at 200X.
(A) Control mice showing normal histoarchitecture, (B) B(a)P
treated mice, (C) Aq.Tc treated mice, (D) B(a)P induced tumor
co-treated with Aq.Tc, (E) AG treated mice, (F) B(a)P induced
tumor co-treated with AG. (EP: extensive proliferation of alveolar
epithelium and presence of hyperchromatic nuclei in the alveolar
wall; SB; small bronchiole, SA; single squamous epithelium,
AW; alveolar wall, AS; alveolar space, AD; Widespread alveolar
destruction, N; thinning of alveolar walls, T; thickening of alveolar
wall, IAS; increased alveolar sac, TB; thickened bronchiolar
epithelium, I; Inflammatory cellular infiltrations.
in B(a)P+AG animals when compared to the B(a)P group
(Table 3).
TUNEL assay
A significant increase in the apoptotic index in B(a)P +
Aq.Tc group was observed when compared with rest of the
four groups i.e., control, AG, B(a)P and Aq.Tc. There was
a significant increase in extent of apoptosis in B(a)P + AG
group but much less than the B(a)P + Aq.Tc group (Figure
6A,B).
Discussion
Lung and the associated respiratory system is the prime
target of the inhaled contaminated air which include
pollutants and carcinogens like B(a)P that have been
established as important etiological agents in causation of
lung cancers (39). Majority of lung cancers are diagnosed
at late stages when the options for treatment are mostly
palliative (40). Management of lung cancer requires
monitoring of the tumor burden to determine the response
to treatment and therefore, improved biomarkers are
A
C
EF
D
B
C
E F
D
A B
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Anticancer eects of Tinospora cordifolia in lung cancer
needed (41). The present study was aimed to evaluate the
efficacy of Aq.Tc extract and its active component, AG, in
B(a)P induced lung tumorigenesis and to demonstrate the
effect on various plasma/serum markers at different stages
of pulmonary carcinogenesis.
To assess the chemopreventive efficacy of Aq.Tc/AG in in
vivo system various parameters were studied in Balb/c mice
pulmonary tumor model. The considerable weight loss
observed in tumor bearing mice could be related to cancer
cachexia, anorexia or malabsorption, which resulted in
wasting of skeletal muscle and adipose tissue (42). Several
studies have shown similar observations of decrease in
body weight of animals following administration of B(a)
P (43). However, Aq.Tc and AG seem to prevent this
alteration in body weight of lung tumor bearing mice.
Chemopreventive response of Aq.Tc and AG administered
animals challenged with B(a)P induced carcinogenesis was
monitored on the basis of tumor incidence, multiplicity,
size and gross morphology. All these parameters serve
as a valuable reference to study the development of
carcinogenesis in in vivo studies. Aq.Tc was successful
in reducing the extent of carcinogenesis as depicted by
different parameters like tumor morphology, multiplicity
and incidence when compared to B(a)P group. Mean
number of large and small tumors was also observed to be
significantly altered in Aq.Tc treated animals. However, in
B(a)P+AG mice less reduction in mean number of small
tumors was observed but the number of large tumors
varied, significantly. Earlier studies also supported our
observation depicting that various plant extracts were
successful in reducing lung adenoma incidence and
multiplicity induced by B(a)P in mice. To the best of our
knowledge no reports are available of chemopreventive
effects of Aq.Tc extract and AG in in vivo lung tumor
model.
Mode of cell death can be a useful parameter to assess the
severity of the cancer (44). Dysregulation of apoptosis is
pivotal to tumorigenesis and the development of most
cancers (45). Aq.Tc treated animals bearing tumors
revealed enhanced apoptosis in comparison to the B(a)
P treated mice bearing pulmonary tumors. Increase in
apoptotic index may also be responsible for the decrease
in the size of the tumors in the B(a)P+Aq.Tc group. The
induction of apoptosis by Aq.Tc extract has already been
documented in the literature as a potential candidate in
therapy to glioblastomas (46). Pure active component AG
of T. cordifolia was found to be less effective in inducing
apoptosis in lung tumor bearing mice. Our lab also
reported the induction of apoptosis by using different
plant extracts in cancer models (47).
Pro-inflammatory cytokines such as TNF-α are the
major molecular players involved in inflammation to
cancer axis. As a representative inflammatory cytokine
with pleiotropic functions, TNF-α plays a dual role in
carcinogenesis (48). In the present study, the gradual
increased levels of TNF-α at 10th and 22nd week of
treatment in lung cancer bearing animals may be due to
pronounced inflammatory response and increased cell
Table 3. Effect of Aq.Tc and AG on various blood tumor markers at different time intervals during B(a)P induced lung tumorigenesis
Parameters/Groups Weeks Control Aq.Tc AG B(a)P B(a)P+Aq.Tc B(a)P+AG
CEA (pg/ml) 0 160.5± 1.56 160.3±9.30 164.5±6.82 159.5±1.36 162.2±10.7 162.9±6.20
10 164.8± 4.54 146.8±12.9 168.1±11.73 251.1±8.64 a1b1c1165.5±11.6 d1
205.3±11.0
c3e3
22 164.2±5.49 176.0±15.4 174±6.57 288.3±21.0
a3b1c1
143.1±16.4
d1
228.7±2.12
b2d2
ctDNA (ng/ml) 0 1.28±0.012 1.11±0.013 1.21±0.010 1.15±0.012 1.17±0.010 1.19±0.020
10 1.29±0.015 1.27±0.062 1.34±0.219 2.95±0.060
a1b1c1
1.65±0.189
a1b1c1d1
2.37±0.210
a1b1c1d2
22 1.41±0.010 1.30±0.010 1.31±0.005 3.28±0.140
a1b1c1
2.33±0.100
d1
2.52±0.136
a1b1c1d2e2
LDH(IU/L) 0 249.5±58.8 304.0±33.9 278.1±45.6 259.1±54.1 259.9±53.0 252.1±51.9
10 233.9±63.8 311.8±46.7 278.1±50.7 688.7±50.7
a1b1c1
465.2±62.5
a2b2c2d2
491.2±56.2
a2b2c2d2
22 246.9±67.2 218.3±48.6 155.4±41.2 628.9±16.5
a2b2c1
293.7±16.2
d3
343.3±27.0
d3
TNF-α (pg/ml) 0 44.6±0.01 46.1±0.01 48.2±2.99 45.4±2.30 44.0±1.70 46.4±2.83
10 46.0±2.35 46.3±2.71 43.6±2.02 59.6±2.91
a1b1c1
49.6±2.04
c1d1
49.4±1.55
c2d1
22 44.6±2.64 46.2±2.71 48.2±2.11 66.9±4.16
a1b1c1
49.8±0.20
d1
51.2±1.44
a3d1
Data is expressed as Mean ± SD (n = 6-8). Data is analysed using one-way ANOVA followed by post-hoc test. a1 P ≤ 0.001, a2 P ≤ 0.01, a3 P ≤ 0.05
signicant with respect to control group; b1 P ≤ 0.001, b2 P ≤0.01 signicant with respect to Tc group; c1 P ≤ 0.001, c2 P ≤ 0.01, c3 P ≤ 0.05 signicant with
respect to Arb group; d1 P ≤ 0.001, d2 P ≤ 0.01, signicant with respect to B(a)P group; e2 P ≤0.01, e3 P ≤ 0.05 signicant with respect to B(a)P+ Tc group.
Journal of Herbmed Pharmacology, Volume 7, Number 4, October 2018 http://www.herbmedpharmacol.com
232
Mohan and Koul
death by B(a)P administration. Also, there is increasing
evidence that TNF-α is mainly produced by cancers and
act as an endogenous tumor promoter (49). Studies have
shown that B(a)P administration promotes the increase of
TNF-α levels in pulmonary tumor bearing mice (50). At
10th week of Aq.Tc and AG treatment to cancer bearing
animals significantly downregulated the levels of TNF-α to
near normal values. The results of the present study are in
collaboration with several other reports which suggested
that T. cordifolia administration to cancer bearing mice
downregulated the levels of TNF-α suggesting the
Figure 6. (A) TUNEL assay performed in paraffin embedded lung/
tumor tissue sections at 200X.
a. Control, b. Benzo(a)pyrene, c. Tinospora cordifolia extract,
d. Benzo(a)pyrene+ Tinospora cordifolia extract group, e. AG
treated, f. Benzo(a)pyrene+ AG treated group.
Green stained cells represent non-apoptotic cells (n), brown
stained cells represent apoptotic cells (a). (B) Attenuation
in apoptotic index of B(a)P induced lung tumors and its
chemoprevention by Aq.Tc and AG.
Data is represented as mean ± SD (n= 5) and analysed by one-
way ANOVA followed by the post hoc test. Statistical significance
and symbols: a1 P ≤ 0.001 w.rt control group; b1 P ≤0.001 w.rt B(a)
P group; c1 P ≤ 0.001 w.rt Aq.Tc group.
0
10
20
30
40
50
60
70
80
Control Aq.Tc AG B(a)P B(a)P+Aq.Tc B(a)P+AG
Apoptotic index
Groups
a1b1c1d1
a1b1c1d1e1
E
C
AB
D
F
immunomodulating activity of T. cordifolia extract (51).
Surprisingly, there was no change in TNF-α levels after
10th week of the treatment protocol in B(a)P +Aq.Tc and
B(a)P +AG treated groups which could be because of
higher apoptotic index found in both these groups.
LDH is a sensitive marker for solid tumors and enhanced
activity of this enzyme has been reported in serum
of various lung tumor patients (52). Serum LDH was
observed to be increased at the promotion stage (10th
week) followed by progression stage (22nd week) of
tumorigenesis in B(a)P treated mice which could be
due to the increased glycolysis in cancerous conditions
or the cellular leakage of these enzymes in circulation.
Our results are in agreement with earlier findings which
depicted that B(a)P administration increased the levels
of LDH in cancer bearing animals (53). A subsequent
decrease in LDH activity on treatment with Aq.Tc aids in
the protection against abnormal cell growth by changing
the permeability of membrane or by affecting the cellular
growth of the tumor. Our lab also reported, reduction of
pulmonary and serum levels of LDH upon administration
of Aloe vera gel extract to cigarette smoke exposed mice
(54). In the present study, AG administration was less
efficient in normalising the LDH activity when compared
to the Aq.Tc extract. AG was found to be less effective
in protecting the membrane permeability and cellular
proliferation of cells damaged by B(a)P insult.
CEA is one of the extensively studied tumor markers
which has also shown prognostic value and has been found
to overexpress on the cell surface of malignant epithelial-
type (55). It is considered as a sensitive tumor marker
for diagnosis, prognosis, and therapy monitoring of lung
cancer (56). Gradual increase in serum CEA levels (10th
and 22nd week) in B(a)P installed animals was observed
which could be associated with increase in tumor growth
and cellular proliferation rate. Available literature also
suggests that CEA levels increase concomitantly in lung
tumor bearing mice (53). Reduction in levels of CEA in
Aq.tc and AG treated animals could be due to decrease
in tumor growth that reveal the anti-tumor effects of
these compounds. Reduction in levels of CEA was more
pronounced in B(a)P + Aq.Tc than B(a)P + AG group
which shows Aq.Tc extract has more potent anticancer
activity. An earlier finding suggested that thymoquinone,
which is the main active constituent of black seed and
essential oil, reduced the serum CEA levels in benzo(a)
pyrene induced lung cancer (57).
Evaluation of plasma ctDNA is a promising diagnostic and
prognostic marker which circumvents the complications
associated with use of invasive techniques used for
biological sampling (58). In the present study, a spike
in ctDNA concentration was prominent at 10th week of
the treatment with B(a)P administration which could
be because of the increase in necrosis of tumor cells, or
release of the intact cells into the bloodstream and their
Journal of Herbmed Pharmacology, Volume 7, Number 4, October 2018
http://www.herbmedpharmacol.com 233
Anticancer eects of Tinospora cordifolia in lung cancer
lysis. Further increase in ctDNA in lung tumor bearing
animals at 22nd week of the treatment could be because
of the severe progression of the tumor cells or the increase
of micro metastatic cells in the blood, which can also shed
their DNA into the blood circulation (59). A significant
drop in ctDNA level was visible in Aq.Tc treated animals
which almost reached the level observed in normal
healthy animals. Also, in animals treated with AG, the
drop in ctDNA level was not as striking as compared to
Aq.Tc treated animals bearing pulmonary tumors. This
decrease in DNA level is either because plasma DNA is
released at lower rates or is due to rapid degrade. ctDNA
release in blood stream could be also correlated to the
mode of cell death as its concentration depends whether
the cell is undergoing apoptotic cell death or necrotic
cell death (60). Histopathological findings were also in
concordance with altered levels of CEA, LDH, ctDNA and
TNF-α, which showed profound increase in their levels at
different stages of carcinogenesis.
Conclusion: Our findings suggest that a concaminant
increase in various tumor markers like CEA, ctDNA,
LDH and TNF-alpha at different stages of carcinogenesis
correlates well with the histopathological changes observed
at early and late stages of B(a)P induced lung cancer.
Aq.Tc successfully improved the various blood markers
and decreased the severity of the disease as revealed by
histopathological and other parameters. Although AG
modulated the above mentioned parameters but Aq.Tc
seemed to be much more effective as a chemopreventive
agent. The histopathological findings in the present study
are in concordance with the biochemical observations
and are suggestive of the fact that Aq.Tc and AG have
a potential as chemopreventive agents in pulmonary
carcinogenesis.
Acknowledgement
We express our heartfelt gratitude to the Department
of Biophysics, Panjab University, Chandigarh, India for
providing the necessary facilities and assistance to carry
out this research work.
Authors’ contributions
The idea of the study, its design and interpretation to
come to a conclusion were done by AK and VM. However,
the experimental procedures were done by VM under the
guidance of AK. Both contribute in preparation of the
manuscript and agreed publication of the final proof.
Conflict of interests
Authors declare no conflict of interests.
Ethical considerations
Ethical issues have been observed by the authors. The
experimental animal studies were approved by the
ethical committee, Panjab University, Chandigarh, India
(Approval no PU/IAEC/S/15/61 Panjab University,
Chandigarh) and were conducted adhering to the Indian
National Science Academy Guidelines for the use and care
of experimental studies.
Funding/Support
This research was financially supported by the University
Grants Commission (UGC) BSR fellowship, New Delhi
(India).
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