Triphala inhibits both in vitro and in vivo xenograft growth of

					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
             Ayurvedic Medicine
•   A system of traditional medicine native to India
•   Ayurveda = “Science of Life”
•   Considered an alternative form of medicine among
    Western contemporaries
•   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
    in mice
•   Suppress the growth of MCF-7 breast cancer cells and protect against radiation
    oxidative-induced damage.
•   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-
     polymerase): mediates
     repair of single strand
     breaks of DNA via activation
     and recruitment of DNA
     repair enzymes

 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
                                  with TPL.
                              •   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
        by Triphala
•    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
                                       antioxidant N-Acetyl
                                       Cysteine (NAC)
                                       inhibited activation of
                                   •   NAC inhibits induced
                                       cell apoptosis due to
                                       lack of histone-
                                       associated DNA
                                       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
      pancreatic ductal
      epithelial cells

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)
                                or TPL.
                            •   Both TPL group showed
                                significantly decreased tumor
                                volume size
                            •   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
                                treated groups.
                        TPL plays a role in activation of induced
                        apoptosis of cancerous pancreatic
                              TUNEL Assay
•   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.

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