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Vol : No. XXVII (4) April, May, June – 2008
Inhibition of in vitro cytotoxic effect evoked by
Alpinia galanga and Alpinia officinarum on PC - 3 cell line
S.Suja* and P.Chinnaswamy**
*PG and Research Department of Biochemistry,
Dr. N.G.P Arts and Science College, Coimbatore-35
**Director Institute of Laboratory Medicines,
Kovai Medical Centre and Research Institute, Coimbatore - 35
mail id- suja_ngp@yahoo.co.in
Received : 12.12.2007 Accepted : 18.02.2008
Abstract :
Plants have been a source of medicine and a major resource for health care since ancient times,
with some traditional herbal medicines having been in use for more than 2,000 years. Herbs
and spices are recommended for prevention and cure of various diseases including cancer.
Alpinia galanga and Alpinia officinarum, botanical cousin to ginger was recognized superior in
many ways and has been employed in medicine for over a thousand years. Prostate cancer is
the most common form of cancer in males and the second leading cause of cancer related death.
PC-3 cell line was derived from adenocarcinoma of human prostate. This was assayed for MTT
assay on treatment with ethanolic extract of Alpinia galanga and Alpinia officinarum, where in
inhibition of the cell growth was noticed. This study was supported by DNA fragmentation
where a characteristic DNA laddering was noticed in treated tumor cell line and not in the
control.
Keywords : Cytotoxicity; MTT assay; Alpinia galanga; Alpinia officinarum ; DNA fragmentation.
Introduction
The human prostate gland, a male sexual
accessory tissue involved in seminal fluid
production, has a remarkably high
incidence of neoplastic disease. Prostate
cancer remains the most common noncutaneous
malignancy in the developed
world and is the second-highest cause of
cancer death in males1. As local prostate
cancer rarely causes symptoms, 38 %-51 %
of patients present with locally extensive or
metastatic disease at the time of diagnosis.
Between 10 % and 50 % of clinically
localized cases inevitably progress and the
patients die from metastatic disease2,3.
Prostate cancer mortality results from
metastases to the bones and lymph nodes
and progression from androgen-dependent
to androgen-independent disease. Although
androgen ablation was found to be effective
in treating androgen-dependent prostate
cancer, no effective life-prolonging therapy
is available for androgen-independent
cancer4. Therefore the androgen
independent cell line (PC3) have been used
as a model for the present work.
A wide number of traditionally important
medicinal plants are still used by Indian
traditional practitioners for the treatment of
cancer. Herbal drugs play an important role
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in health care programmes world wide, and
there is a resurgence of interest in herbal
medicines for treatment of various ailments
including cancer. Medicinal herbs and
extracts prepared from them are widely
used in the treatment of liver diseases like
hepatitis, cirrhosis, and loss of appetite 5.
Alpinia galanga (greater galanga) and Alpinia
officinarum (lesser galangal) are ancient and
highly revered medicinal agent in
Ayurvedic, Traditional Chinese and Thai
folk medicine. This botanical cousin to
ginger was recognized to be superior in
many ways and has been employed in
medicine for over a thousand years. It has a
pungent, hot and spicy taste with an
aromatic, ginger-like odor. In traditional
Chinese Medicine it is said as “warm the
middle” and alleviate pain, being especially
good for abdominal pain, vomiting, hiccups
and diarrhea 6.It is also recognized as a
powerful dispersing agent with analgesic
properties that make it an appropriate and
effective intervention for rheumatic pains
and other inflammatory disorders 7.The
present study was thus designed to find the
anticancer effect of Alpinia galanga and
Alpinia officinarum against prostate cancer
cell line (PC3).
Materials and Methods
Collection of plant material: Dried root of
Alpinia galanga and Alpinia officinarum were
collected from Ayurvedic pharmacy,
Coimbatore and authenticated by the
botanist Dr.
Arumugaswamy, Department of Botany,
Kongunadu college of arts and science,
Coimbatore, and the voucher specimen was
preserved in Department of Biochemistry,
Dr N.G.P Arts and Science college,
Coimbatore, India.
Preparation of Plant extract: Dried root of
Alpinia galanga and Alpinia officinarum were
made into coarse powder using mixer
grinder. The powder obtained was
successfully extracted in petroleum ether
(60-800c), chlororoform, ethanol and
distilled water by using soxhlet extractor.
The extracts were concentrated in a rotar
evaporator under reduced pressure. A pilot
study revealed that ethanolic extract evoked
the maximum activity. It has been found
that IC50 value was noted to be 37.6 for
Alpinia galanga, and 41.45 for Alpinia
officinarum.
Cell culture PC-3, derived from
adenocarcinoma of human prostate, was
purchased from the National Centre for Cell
Sciences, Pune, India. The cells were grown
in Ham’s F12K medium with 2 mM Lglutamine
adjusted to contain 1.5 g/liter
sodium bicarbonate (90%) supplemented
with 10% fetal bovine serum, as
recommended by American Type Culture
Collection. The cells were maintained at
37°C in a humidified atmosphere of 95% air
and 5% CO2..
Preparing Cancer Cell Lines: Prostate Cancer
Cell Line (PC-3) was grown at 37° C. in Ham’s
F12K Media. The cells were grown until
confluent and then washed once with PBS and
aspirated. The cells were then washed in
Trypsin, Ham’s F12K medium was added, and
the cell solution was aspirated in the test tube
and centrifuged for 5 minutes at 6000 rpm. After
centrifugation, the supernatant was discarded
and the pellet was resuspended in 2 ml of Ham’s
F12K medium . 0.5 ml of this solution was
combined with 15 ml of Ham’s F12K medium
and placed in a cell culture tube. The cells were
counted by placing 20 μl of the cell suspension
in a Hemocytometer, diluting as necessary for
calculation. The cell concentration was kept at
approximately ten thousand per μl for
testing against the extracted compounds.
MTT Assay: The cells were seeded in 96
well plate(1X106 cells/well) and allowed to
attach by incubating at 370C for 24 hours.
The cells were treated with the drug (10,
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25, 50, 100 μg of the ethanolic extract of
Alpinia galangal and Alpinia officinarum
respectively) and incubated the wells for
24hrs. The medium was removed and 100
μl of MTT reagent added to the well, and
after incubation for 24hrs at 37°C, the
medium was removed completely. 100 μl of
SDS has then added to each well and
incubated at 370C for 4 hours, the well
plates were read at 570nm using ELISA
reader. Each assay was performed in
triplicate8.
DNA Fragmentation Analysis., PC-3 cells
were seeded in 6 well plates and allowed to
adhere overnight. After treatment with the durg (
ethanolic extract of Alpinia galanga and Alpinia
officinarum) respectively as per the same
schedule described above, cells were harvested
and then lysed in a solution containing 100 mM
NaCl, 10 mM Tris (pH 7.4), 25 mM EDTA, and
0.5% SDS. After the centrifugation, the
supernatants were incubated with 300 mg/ml
proteinase K for 5 h at 65°C and extracted with
phenol-chloroform. The aqueous layer was
treated with 0.1 volume of 3 M sodium acetate,
and the DNA was precipitated with 2.5 volumes
of 95% ethanol. After treatment with 100 mg/ml
RNase A for 1 h at 37°C, the sample was
electrophoresed on a 2% agarose gel and stained
with ethidium bromide9.
Results and Discussion
Cancer has been scourging on the human
population since long. Although numerous
advances have been made in prevention,
diagnosis and treatment of the disease, it
still continues to torment mankind 10, 11.
Foods rich in antioxidants have been shown
to play an essential role in the prevention of
cardiovascular diseases, cancer, neurodegerative
diseases, inflammation and problems caused by
cell and cutaneous aging12.
Cell viability (MTT) Assay
S.No Concentration of Percentage inhibition
Extract in( μg) Alpinia galanga Alpinia officinarum
1. 10 28±2.36 25 ± 1.46
2. 25 44± 1.53 41 ± 3.15
3. 50 55±1.82 52 ± 2.63
4. 100 69±3.91 63 ± 2.80
Values represented as mean ± SD(N=3)
Fig 1 MTT CYTOTOXICITY ASSAY OF
Alpinia galanga
Fig 2 MTT CYTOTOXICIRY ASSAY OF
Alpinia officinarum
0
10
20
30
40
50
60
70
80
0 20 40 60 80 100 120 Co nc e ntratio n o f Extrac t in μg / ml
Cell Viability
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Fig 2 MTT CYTOTOXICIRY ASSAY OF
Alpinia officinarum
4.5.2 DNA Fragmentation Assay
The concentration of the ethanolic extract of
Alpinia galanga and Alpinia officinarum
which showed 50 % inhibition of Tumor cell
line growth as obtained from the MTT assay
was used for the DNA fragmentation assay.
Fragmentation of genomic DNA to high
molecular weight (180kb-200kb) fragments
is a characteristic of the early event in
apoptosis and may represent the key step of
the process. Apoptosis or programmed cell
death represents a physiological form of cell
death that occurs during development and
in the mature animal.
Agarose gel electrophoresis was used to
evaluate whether high molecular DNA
fragmentation was a feature of the cellular
response of the tumor cell to the ethanolic
extract of Alpinia galanga and Alpinia
officinarum. The internucleosomal DNA
was detected on agarose gel as a ladder of
DNA fragment (figure 2) and this
confirmed that the cytotoxic effect of the
Alpinia extracts were mediated via
apoptosis. It is emphasized that there was a
close relationship between apoptosis and
proliferation in malignant neoplasm cells. A
characteristic DNA laddering was noticed
only in tumor cell line treated with the
ethanolic extract of Alpinia galanga and
Alpinia officinarum. This pattern was not
observed in the control cells. The data
reported here demonstrated the lethal
injury to carcinoma cells via activation of
the cell death by both the ethanolic extract
of Alpinia galanga and Alpinia officinarum
(fig3).
Emerging research suggests that Alpinia
extracts may have potent anticancer activity, for
both breast and prostate cancer. Research
shows that 30 to 35% of all cancers can be
prevented by eating well, being active and
maintaining a healthy body weight
(Canadian Cancer Society). However, much
more research, including human studies,
needs to be done before any specific
recommendations can be made.
The specificity of an anticancer compound
against cancer cell depends on the
proliferation rates of the treated cells.
Cancer cells proliferate more rapidly than
normal cells, which make them more
sensitive to drug treatment. Plant-derived
anticancer agents act by entering with
cancer cell proliferation or by the induction
of apoptosis in tumor cells13. The advantage
of apoptosis inducing anticancer drugs is
that the apoptotic bodies formed can be
0
10
20
30
40
50
60
70
80
0 20 40 60 80 100 120
Co nc e ntratio n o f Extrac t in μg / ml
Cell Viability
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scavenged by the human body’s immune
system without inducing an inflammatory
response14. In addition to apoptosis
induction, another common effect shared by
many anticancer drugs is the introduction
of cell cycle arrest. Cell cycle arrest has been
found in cells exposed to a variety of
stimuli, including irradiation, microtubule
stabliling agents and topoisomerase
inhibitor 15. .Lee C.C. & Houghton P. (2005)
has established that extracts from A.
officinarum can exhibit cytotoxicity against
human cancer cell-lines16
Fig. 3 GEL Electrophoresis
Based on the above study the ethanolic
extract of Alpinia galanga and Alpinia
officinarum were able to inhibit the growth
and induce apoptosis of cancer cells invitro.
Conclusion
The MTT analysis carried out in the prostate
cancer cell line (PC-3) revealed that the
ethanolic extract of Alpinia galanga and
Alpinia officinarum could effectively reduce
the growth and multiplication of the tumor
cells and ultimately suppress the growth of
the tumor cells. This study was supported
by DNA fragmentation analysis which
proved that characteristic DNA laddering
was noticed only in tumor cell line treated
with the ethanolic extract of Alpinia galanga
and Alpinia officinarum. This pattern was not
observed in the control cells. The
anticarcinogenic effect of the ethanolic
extract of Alpinia galanga and Alpinia
officinarum may be due to rich antioxidant
content and the presence of secondary
metabolites like flavonoids present in them.
Lane 1 2 3 4 5 6
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