Triphala inhibits both in vitro and in
vivo xenograft growth of pancreatic
tumor cells by inducing apoptosis
Yan Shi, Ravi P Sahu and Sanjay K
Presented by John Campiche & Kelsey Roe
• A system of traditional medicine native to India
• Ayurveda = “Science of Life”
• Considered an alternative form of medicine among
• Complementarily administered system involving: herbs,
massage and yoga
• Basic doctrine of Ayurvedic medicine focuses on building a
healthy metabolic system, employing proper elimination
of wastes and maintaining good digestion
• These cultural practices have led to a number of medicinal
preparations and surgical procedures for curing various
ailments and diseases
• Will ultimately lead to vitality and longevity
• Most commonly used Indian Ayurvedic herbal formulation.
• Consisted of three equal parts of medicinal dried fruits
• Major ingredients shown to be gallic acid and ascorbic acid.
• Medicine is rich in natural antioxidants and is believed to promote immunity,
health and longevity
• A great deal of research is being conducted in India to pursue an understanding of
the underlying biochemical mechanisms associated with Triphala of which they
are currently unknown
• Triphala significantly reduced benzo(a)pyrene-induced forestomach tumorigenesis
• Suppress the growth of MCF-7 breast cancer cells and protect against radiation
• Has been shown to provide enhanced cytotoxic effects on cancer cell lines in vitro
Caspase Pathway Leading to
The effects of Triphala on the
survival of human pancreatic
• Capan-2 Cells: Human
pancreatic cancer cells that
express wild-type p53
• PARP (poly-ADP-ribose-
repair of single strand
breaks of DNA via activation
and recruitment of DNA
Triphala induces apoptosis in
human pancreatic cancer cells
with an IC50 of 50µg/mL
Triphala causes DNA damage resulting in activation of
p53 in Capan-2 cells
• Triphala treatment for 24 h led to
phosphorylation of H2A.X at Ser-
139 suggesting the presence of
DNA ds breaks.
• DNA damage leads to activation
of p53 by ATM Kinase
• 1 h treatment showed sig.
upregulation of p53 as well as
downstream component p21.
• Cells treated with pifithrin- α, a
p53 inhibitor, showed inhibition of
activation following treatment
• Pifithrin- α also blocked PARP
cleavage in the presence of TPL.
Suggests TPL provokes an
activation of p53 pathway
Normal Activation of p53 pathway
• negative regulator protein
• binds to N-terminal trans-activation
domain to inhibit activity of p53.
Activation of ERK
• ERK: Part of the MAPK
• Elk: A downstream
substrate of ERK
• MEK: An upstream
regulator of ERK
• U0126: An inhibitor of MEK
apoptosis is mediated by
ERK. ERK may be an
upstream regulator of
p53 in this system.
Triphala-induced ROS generation triggers ERK
activation and apoptosis in Capan-2 cells
• Treatment of Capan-2
cells with 60 ug/ml
showed increased ROS
generation in ½ h
• Treatment with
inhibited activation of
• NAC inhibits induced
cell apoptosis due to
lack of histone-
fragments in the
presence of TPL.
Suggests Triphala mediated ROS is
responsible for activation of ERK
and/or p53 in induced cell apoptosis
The effect of Triphala
is not cell specific
• BxPC-3: Human
pancreatic cancer cell line
that express mutated p53.
• HPDE-6: Normal human
Treatment of BxPC-C cells
with Triphala showed a
reduced survival rate with an
IC50 of 85µg/mL. However
Triphala failed to induce
apoptosis in non-cancerous
Triphala inhibits the growth of Capan-2 human
pancreatic xenografts in vivo
• Pancreatic tumor cells were
implanted in nude mice
through subcutaneous injection
followed by oral uptake of
Phosphate buffered saline(PBS)
• Both TPL group showed
significantly decreased tumor
• TUNEL assay showed increased
number of apoptotic cell bodies
in TPL groups.
• Cleavaged PARP and caspase-3
components were shown in
Western blot analysis of TPL
TPL plays a role in activation of induced
apoptosis of cancerous pancreatic
• Terminal deoxynucleotidyl transferase
dUTP nick end labeling (TUNEL)
QuickTime™ and a
• Determines presence of DNA TIFF (Un compressed) decompressor
are neede d to se e this picture.
fragmentation by labeling terminal
ends of nucleic acids.
• Addition of dUTP catalyzed by
terminal deoxynucleotidyl transferase, dUTP
adds dUTP to 3’ ends of ss or ds DNA.
• dUTP are secondarily labeled to
determine presence of fragments.
QuickTime™ and a
TIFF (Un compressed) decompressor
are neede d to se e this picture.
dUTP + marker
Summary and Conclusions
• Triphala treatment reduces the survival rate of human pancreatic cancer cells in
vitro, but failed to cause cytotoxic effects on non-cancerous cells.
• Triphala induced apoptosis in Capan-2 cells was associated to the generation of
reactive oxygen species.
• The generation of reactive oxygen species caused DNA damage resulting in the
activation of ATM and ERK which lead to the stabilization of p53.
• By introducing U0126, MEK and therefore ERK was inhibited; and Pifithrin-α
inhibited p53 activity in Capan-2 cell.
• U0126 treatment also blocked apoptosis in Triphala treated BxPC-3 cells as well.
This suggested that ERK was a molecular target of Triphala in pancreatic cancer
• Triphala caused reduced tumor growth in vivo. Mice with Capan-2 xenografts were
treated with Triphala every 5 days orally.
• Increased apoptosis of these tumor cells in mice was observed, and was due to the
activation of ERK and p53.