Triphala inhibits both in vitro and in vivo xenograft growth of pancreatic tumor cells by inducing apoptosis

1. 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 Srivastava Presented by John Campiche & Kelsey Roe

2. 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

3. Triphala • 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

4. Caspase Pathway Leading to Apoptosis

5. The effects of Triphala on the survival of human pancreatic cells. • 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

6. 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

7. Normal Activation of p53 pathway • mdm2 • negative regulator protein • binds to N-terminal trans-activation domain to inhibit activity of p53. http://en.wikipedia.org/wiki/P53

8. Activation of ERK by Triphala • ERK: Part of the MAPK pathway • Elk: A downstream substrate of ERK • MEK: An upstream regulator of ERK • U0126: An inhibitor of MEK Triphala-induced apoptosis is mediated by ERK. ERK may be an upstream regulator of p53 in this system.

9. 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 ERK. • 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

10. 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 cells.

11. 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 xenografts

12. TUNEL Assay • Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) • Determines presence of DNA fragmentation by labeling terminal ends of nucleic acids. • Addition of dUTP catalyzed by terminal deoxynucleotidyl transferase, adds dUTP to 3’ ends of ss or ds DNA. • dUTP are secondarily labeled to determine presence of fragments. dUTP dUTP + marker

13. 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 cells. • 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.

14. Questions?