RESEARCH COMMUNICATIONS
Preclinical studies on the use of
medicinal mushroom Ganoderma
lucidum as an adjuvant in
radiotherapy of cancer
G. Gopakumar, Femy Martin,
Sherin K. Antony, Thulasi G. Pillai and
Cherupally Krishnan K. Nair*
Amala Cancer Research Centre, Thrissur 680 555, India
Our previous studies have demonstrated that an
extract of Ganoderma lucidum occurring in South
India possesses significant radioprotective property ex
vivo. The present study describes the in vivo radiopro-
tection of normal cells in tumour-bearing mice
exposed to gamma radiation. Oral administration of
G. lucidum extract (GLE) to tumour-bearing Swiss
albino mice along with exposure to gamma radiation
resulted in tumour regression. Single-cell gel electro-
phoresis (comet assay) on cells of normal and tumour
tissues from tumour-bearing animals treated with
GLE and radiation, revealed that there was significant
reduction in radiation-induced damage to cellular
DNA in normal tissues compared to the tumour, indi-
cating preferential protection to normal tissues. The
findings suggest the potential use of this mushroom
extract as an adjuvant in radiotherapy, for tumour
regression and prevention of radiation-induced cellu-
lar damages in normal tissues.
Keywords: Adjuvant, cancer, Ganoderma lucidum,
preclinical studies, radiotherapy.
IONIZING radiation is one of the well-established and
widely used therapeutic modalities either for curative or
palliative treatment of tumours, but the major problem
associated with cancer radiotherapy is the severe side
effects and damage to normal tissues1. In radiotherapy of
cancer, normal tissues need to be protected whereas cancer
cells are exposed to high radiation. Even though many
compounds have been studied for their radioprotecting
property, an agent producing differential radiation response
in the tumour and normal cells would be of importance in
effective treatment of cancer by radiation therapy2.
Ganoderma (Figure 1), commonly known as reishi
mushroom is highly ranked in Oriental folklore. In Chi-
nese medicine reishi has been considered as a panacea for
all types of diseases. Reishi has attracted significant
attention in recent years due to its large number of phar-
macological properties. The fruiting bodies of this mush-
room contain a variety of chemical substances3. A
number of medicinal mushrooms have recently been
reported to possess significant antioxidant activity. One
*For correspondence. (e-mail: ckknair@yahoo.com)
1084
of the major activities reported for Ganoderma is its anti-
oxidant activity. Ganoderma lucidum extract (GLE) also
contains ergosterols, complete proteins, unsaturated fatty
acids, vitamins and minerals. It is the only known source
of a group of triterpenes known as ganoderic acids, which
have a molecular structure similar to steroid hormones
and contains the most active polysaccharides among me-
dicinal plant sources. Ganoderic acids may lower blood
pressure and decrease LDL cholesterol4. Anti-aging prop-
erties have also been reported for this mushroom. In addi-
tion, it is reported that the mushroom is used for the
preparation of an HIV tonic5. G. lucidum has been found
to play an important role in combination with radiother-
apy and chemotherapy, to render complete regression of
the tumours6–8. Since both polysaccharides and organic
germanium derived from G. lucidum are not cytotoxic
to tumour cells, the anti-tumour effect is attributable to
induced immunopotentiation.
Our earlier reports suggest that the aqueous extract of
this mushroom has significant radioprotective activity ex
vivo9. The present study describes the in vivo radioprotec-
tion of normal cells in tumour-bearing mice exposed to
whole-body gamma radiation and the sensitization of its
tumour to radiation (as there was enhanced radiation-
induced damage in cellular DNA in the tumour) by oral
administration of GLE. The work is also focussed on the
effect of GLE in tumour regression in tumour-bearing
Swiss albino mice when administered orally along with
radiotherapy.
GLE was prepared from G. lucidum collected from the
outskirts of Thrissur, Kerala, India. Sporocarps of the
mushroom were dried at 40–50°C and powdered. Several
batches of 100 g powder were extracted with 1 : 1 etha-
nol : distilled water mixture at 80°C for 8–10 h. The
extracts were combined, filtered, concentrated and evapo-
rated at low temperature. The residue thus obtained was
used for the experiments. The yield was 10.6%.
Swiss albino mice about 8–10 weeks old and weighing
22–25 g were purchased from the Small Animal Breeding
Figure 1. Ganoderma lucidum.
CURRENT SCIENCE, VOL. 99, NO. 8, 25 OCTOBER 2010
 RESEARCH COMMUNICATIONS
Section (SABS), Mannuthy, Thrissur. They were main- Animals in groups I and II were orally administered
tained under standard conditions of temperature and with distilled water, and groups III and IV with
humidity in the Animal House Facility at our Centre. The 200 mg/kg GLE. The animals in groups II and IV were
animals were provided with standard mouse chow (Sai exposed to 4 Gy whole-body gamma radiation, 1 h after
Durga Feeds and Foods, Bangalore) and water ad libitum. administration of distilled water or GLE. Immediately
All animal experiments in this study were carried out after irradiation the animals were sacrificed and blood,
with the prior approval of the Institutional Animal Ethics brain, bone marrow and tumour tissues were collected for
Committee (IAEC) and were conducted strictly adhering performing alkaline single-cell gel electrophoresis (comet
to the guidelines of Committee for the Purpose of Control assay)10,11.
and Supervision of Experiments on Animals (CPCSEA), The DNA strand breaks in various tissues of mice were
constituted by the Animal Welfare Division, Government estimated using alkaline single-cell gel electrophoresis
of India. which was performed using the method given by Singh10,
Na2-EDTA was purchased from Sisco Research Labo- with minor modifications11. Microscopic slides were
ratories Ltd, Mumbai. High melting point agarose, low coated with normal melting point agarose (1% in PBS
melting point agarose, TritonX-100, DMSO and bovine containing 0.8% NaCl, 0.02% KCl, 0.14% Na2HPO4,
serum albumin were purchased from Sigma Chemical 0.02% NaH2PO4), the coverslip was placed immediately
Company Inc, MO, USA. All other chemicals were of and kept at 4°C for 10 min to solidify the agarose. After
analytical grade procured from reputed Indian manufac- removal of the coverslip, 200 μl of 0.8% low melting
turers. point agarose containing 50 μl of treated cells was added
Irradiation, i.e. exposure to gamma radiation was car-
ried out using a 60Co-Theatron Phoenix teletherapy unit
(Atomic Energy Ltd, Ottawa, Canada) at the Amala Can-
cer Hospital, Thrissur at a dose rate of 1.88 Gy/min.
The combined effect of 4 Gy gamma radiation and
GLE on tumour growth in vivo was analysed as follows.
Solid tumour was produced by injecting Dalton’s Lym-
phoma Ascites (DLA) cells (1 × 106 cells/animal) subcu-
taneously into the right hind limb of Swiss albino mice
weighing 22–25 g. The animals were divided into four
groups, each consisting of six animals. The first group
was kept as untreated control, the second received 16
doses of GLE in 15 days (200 mg/kg body wt/dose), the
third was exposed to single dose of 4 Gy gamma radia-
tion and fourth group was administered with 200 mg/kg
body wt of GLE 1 h prior to and immediately after expo-
sure to 4 Gy gamma radiation and also daily for the next
14 days. The treatments were started on the 7th day after
transplanting tumour cells (when the tumour reached
a size of 1.0 cm3) and continued for 15 consecutive days.
The thickness of the hind leg was measured using a
vernier calliper once in three days from the 7th day of
tumour transplantation. The tumour volume was cal-
culated as follows: Tumour thickness = Thickness of
tumour-induced leg – Thickness of normal leg.
Tumour volume = 4/3π r3, where r is the tumour
radius, the average of r1 and r2 which are the tumour
thicknesses.
The effect of GLE on radiation-induced damage in cel-
lular DNA of cells of normal tissues and tumour in Figure 2. Effect of Ganoderma lucidum extract (GLE) and 4 Gy
tumour-bearing mice exposed to whole-body gamma gamma radiation on solid tumour in mice. Swiss albino mice bearing
radiation was studied as follows. Swiss albino mice were solid tumour (Dalton’s Lymphoma Ascites) on hind limbs were orally
administered with GLE and exposed to 4 Gy gamma radiation. After
divided into four groups: Group I – 0.2 ml distilled water radiation exposure, GLE administration was continued for 14 days and
(oral) + Sham irradiation; Group II – 0.2 ml distilled the volume of tumour was monitored for 30 days. Photographs show an
water (oral) + 4 Gy 60Co-γ-rays; Group III – 200 mg/kg animal and its hind limb bearing the tumour on the 1st, 7th and 30th
day following various treatments – day 1 at the start of the treatment,
GLE (oral) + Sham irradiation and Group IV – 200 mg/kg 1a and b – 0 Gy; 2a and b – 0 Gy + GLE; 3a and b – 4 Gy; 4a and b –
GLE (oral) + 4 Gy 60Co-γ-rays. 4 Gy + GLE.

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Figure 3. Effect of GLE and gamma radiation on solid tumour in mice. Swiss albino mice bearing solid tumour
(Dalton’s Lymphoma Ascites) in hind limb were administered with GLE and exposed to 4 Gy gamma radiation.
After radiation exposure, GLE administration was continued for 14 days and the tumour volume monitored on
alternate days, as described in the text.

Figure 4. Photographs of silver-stained comets from cells of different tissues of tumour-bearing Swiss albino mice orally admin-
istered with GLE and exposed to 4 Gy gamma radiation.

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Figure 5. Effect of oral administration of GLE on radiation-induced cellular DNA damage as assessed by comet assay in brain cells of mice-
bearing tumour on hind limbs, exposed to 4 Gy whole-body gamma radiation. Mean comet parameters like %DNA in tail, tail length, tail moment
and Olive tail moment of single cells subjected to alkaline single-cell gel electrophoresis are presented with ± SD. ns, Not significant; ***P < 0.001
when compared with respective control.
to the slides, the coverglasses were placed immediately
and the slides were kept at 4°C. After solidification, the
coverglasses were removed and the slides were immersed
in pre-chilled lysing solution containing 2.5 M NaCl;
100 mM Na2EDTA, 10 mM Tris-HCl (pH-10), 1%
DMSO and 1% TritonX, and kept for 1 h at 4°C. After
lysis, the slides were drained properly and placed in a
horizontal electrophoretic apparatus filled with freshly
prepared electrophoresis buffer containing 300 mM
NaOH, 1 mM EDTA and 0.2% DMSO (pH ≥ 13). The
slides were equilibrated in the buffer for 20 min and elec-
trophoresis was carried out for 30 min at 25 V and
300 mA. After electrophoresis the slides were washed
gently with 0.4 mM Tris-HCl buffer (pH 7.4) to remove
the alkali. The slides were again washed with distilled
water and kept at 37°C for 2 h to dry the gel; then silver
staining was carried out11. The comets were visualized
using Olympus BX-41 microscope and the images were
captured and analysed using the software ‘CASP’, which
directly gave the comet parameters such as %DNA in tail,
tail length, tail moment (TM) and Olive tail moment
(OTM)12. TM is the product of tail length and %DNA in
tail, and OTM is the product of the distance between the
centre of gravity of the head and centre of gravity of the
tail and %DNA in the tail10–12. Data of the comet para-
meters were presented as mean ± standard deviation.
CURRENT SCIENCE, VOL. 99, NO. 8, 25 OCTOBER 2010
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The results of the study on the effect of gamma radia-
tion (4 Gy) and GLE administration are presented in Fig-
ures 2 and 3. Figure 3 presents data on tumour volume,
following the treatments and Figure 2 shows representa-
tive photographs of the animal and its tumour-bearing
limb. The growth of the tumour was found to be inhibited
in animals exposed to 4 Gy gamma radiation compared to
untreated animals. The GLE itself reduced tumour growth
to some extent. The tumour growth was found substan-
tially inhibited in the group of animals administered with
GLE and exposed to 4 Gy gamma radiation.
Alkaline comet assay was performed to analyse the
effect of administration of GLE on radiation-induced cel-
lular DNA damage in normal and tumour tissues. Figure
4 shows representative photographs of the comets from
the cells of these tissues. It can be seen from Figures 5–8
that the cellular DNA from various tissues such as blood,
bone marrow, brain and tumour of the tumour-bearing
animals exposed to whole-body 4 Gy gamma radiation
shows increased comet parameters such as %DNA in tail,
tail length, TM and OTM. However, except in the tumour
tissues, these parameters were found to be lower in the
normal tissues of tumour-bearing animals administered
with GLE 1 h prior to radiation exposure (Figures 5–8).
This would suggest that the administration of GLE
offered protection against radiation-induced damages to
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Figure 6. Effect of oral administration of GLE on radiation-induced cellular DNA damage as assessed by comet assay in bone marrow cells of
mice-bearing tumour on hind limbs, exposed to 4 Gy whole-body gamma radiation. Mean comet parameters like %DNA in tail, tail length, tail
moment and Olive tail moment of single cells subjected to alkaline single-cell gel electrophoresis are presented with ± SD. ns, Not significant;
***P < 0.001 when compared with respective control.

Figure 7. Effect of oral administration of GLE on radiation-induced cellular DNA damage as assessed by comet assay in blood leucocytes of
mice-bearing tumour on hind limbs, exposed to 4 Gy whole-body gamma radiation. Mean comet parameters like %DNA in tail, tail length, tail
moment and Olive tail moment of single cells subjected to alkaline single-cell gel electrophoresis are presented with ± SD. ns, Not significant;
***P < 0.001 when compared with respective control.

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Figure 8. Effect of oral administration of GLE on radiation-induced cellular DNA damage as assessed by comet assay in tumour cells of mice-
bearing tumour on hind limbs, exposed to 4 Gy whole-body gamma radiation. Mean comet parameters like %DNA in tail, tail length, tail moment
and Olive tail moment of single cells subjected to alkaline single-cell gel electrophoresis are presented with ± SD. ns, Not significant; ***P < 0.001
when compared with respective control.
cellular DNA in normal tissues, and in the tumour tissues
the extract offered no protection but helped enhance the
radiation-induced cellular DNA damage, as can be evi-
denced from the data in Figures 4–8.
The present study is focused on the radioprotective and
anti-cancer properties of G. lucidum under in vivo condi-
tions using mouse as the model system. Radioprotective
agents offer a possible solution to counteract the radiation
damage to living systems1. The extracts of G. lucidum,
certain medicinal plants, vitamin derivatives and dietary
supplement formulations with good anti-oxidant activity
can be considered as safe radioprotectors, whereas many
of the synthetic drugs and chemicals prepared for radio-
protection have limited application in the living systems
due to their toxicity and side effects13–17. Our previous
studies have revealed that aqueous extract of G. lucidum
possesses radioprotective activity9.
GLE has been reported to exhibit anti-tumour activity
and this has been attributed to immune-related mecha-
nisms or cytotoxicity18–20. Some of the active ingredients
in the extract have been identified as polysaccharides and
triterpenes21,22. The extract has also been shown to pre-
vent proliferation of cancer cells, mediated through inhi-
bition of DNA synthesis23. Inhibition of DNA synthesis
following irradiation could bring about enhanced apop-
tosis in the cells. This could be a possible mechanism by
which GLE enhances the anti-tumour activity of gamma
radiation.
The tumour regression study carried out with solid
tumour-bearing animals revealed that oral administration
CURRENT SCIENCE, VOL. 99, NO. 8, 25 OCTOBER 2010
RESEARCH COMMUNICATIONS
of GLE (200 mg/kg body wt) to the animals resulted in sig-
nificant reduction in tumour volume, and this anti-tumour
effect was more prominent in conjunction with gamma-
radiation treatment (4 Gy). Studies on in vivo radiopro-
tection efficiency by comet analysis demonstrated the
efficiency of GLE to offer protection to normal tissues
against gamma radiation-induced DNA damage, whereas
sparing tumour tissues where the extract offered no pro-
tection against radiation-induced cellular DNA damage.
The mushroom G. lucidum could be effectively used as
a radioprotector for normal tissues in radiotherapy. It is to
be noted that there was an increased extent of cellular
DNA damage in cells of the tumour tissues of animals
administered with GLE prior to radiation exposure which
could be due to induction of apoptosis in these cells. Fur-
ther studies are needed to support this possibility. The
present results suggest the possibility of using this me-
dicinal mushroom extract as an adjuvant in cancer radio-
therapy to protect normal tissues from radiation damage
and also to enhance the anti-tumour activity of gamma
radiation.
1. Nair, C. K. K., Parida, D. K. and Nomura, T., Radiation protectors
in radiotherapy. J. Radiat. Res., 2001, 42, 21–37.
2. Shueng, P. W., Hsu, W. L., Jen, Y. M., Wu, C. J. and Liu, H. S.,
Neoadjuvant chemotherapy followed by radiotherapy should not
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Radiat. Oncol. Biol. Phys., 1998, 40, 889–896.
3. Tim, L., Yihuai, G. and Shufeng, Z., Global marketing of medici-
nal Ling Zhi mushroom G. lucidum products and safety concerns.
Int. J. Med. Mushroom, 2004, 6, 189–194.
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4. Maruyama, H. K. and Murofushi, Antitumour activity of Sarcodon
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5. Chang, R. T., Limitations and potential applications of Gano-
derma and related fungal polyglycans in clinical oncology. In First
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6. Wang, D. H. and Weng, X. C., Antitumour activity of extracts of
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9. Pillai, T. G., Salvi, V. P., Maurya, D. K., Nair, C. K. K. and
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of India. Curr. Sci., 2006, 91(3), 341–344.
10. Singh, N. P., Microgels for estimation of DNA strand breaks,
DNA – protein cross links and apoptosis. Mutat. Res., 2000, 455,
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11. Chandrasekharan, D. K., Kagiya, V. T. and Nair, C. K. K., Radia-
tion protection by 6-palmitoyl ascorbic acid-2-glucoside: studies
on DNA damage in vitro, ex vivo, in vivo and oxidative stress in
vivo. J. Radiat. Res., 2009, 50, 203–212.
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sis programme for the comet assay. Mutat. Res., 2003, 534, 15–20.
13. Weiss, J. F. and Landauer, M. R., Protection against ionizing
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and future prospects. Phytother. Res., 2005, 19, 1–22.
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Murase, H., Gu, Y. H. and Kagiya, T. V., Water soluble vitamin E
(TMG) as a radioprotector. Indian J. Exp. Biol., 2003, 41, 1365–
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and Lin, C. H., Studies on the immuno-modulating and antitumour
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ACKNOWLEDGEMENT. We thank the DST, New Delhi for finan-
cial support through research grant given to C.K.K.N.
Received 17 August 2009; revised accepted 31 August 2010
DNA microarray analysis targeting
pmoA gene reveals diverse community
of methanotrophs in the rhizosphere of
tropical rice soils
Pranjali Vishwakarma and Suresh K. Dubey*
Department of Botany, Banaras Hindu University,
Varanasi 221 005, India
The diversity of the methanotrophs community of two
different rice fields of a typical tropical rice agroecosys-
tem was assessed using microarray targeting pmoA
gene-based approach. The presence of types I and II
methanotrophs was observed with the dominance of
Methylocystis in both the fields. The study revealed that
the Barkachcha rice field harbours more diverse groups
of methanotrophs than the Ghazipur rice field. It was
also observed that in some members of types I and II
methanotrophs, even the peat-associated group was
present in the enriched culture of the soils. The
Ghazipur soil and its enriched mixed methanotrophic
culture showed higher methane oxidation potential
than the Barkachcha soil. These results suggest that the
methanotrophs community and its potential for methane
oxidation vary with change in soil type within the
same ecosystem.
Keywords. Methane oxidation, methanotrophs, micro-
array, pmoA gene, rice soil.
DUE to their significant role in global CH4 cycling, meth-
ane-oxidizing bacteria (MOB; methanotrophs) have been
the focus of several scientific researchers. Methanotrophs,
abundantly found in the aerobic layer of the soil, the
rhizosphere1,2, oxidize significant amount of CH4 gener-
ated by methanogens. It is expected that methanotrophic
bacteria utilizing CH4 as substrate in the rhizosphere will
vary in the population composition and density within the
rice rhizosphere3. The methanotrophic community is
complex and diverse, containing 10 genera which belong
to type I (Gammaproteobacteria), and four genera to group
*For correspondence. (e-mail: skdubey@bhu.ac.in)
CURRENT SCIENCE, VOL. 99, NO. 8, 25 OCTOBER 2010