Triphala has been used for centuries in Ayurvedic medicine to treat various types of gastrointestinal-related disorders; however, the molecular mechanisms of Triphala have not been studied yet. In the present studies, we demonstrate that aqueous extract of Triphala is effective in inhibiting the growth of pancreatic cancer cells in culture as well as in the in vivo model. Our results reveal that Triphala treatment drastically reduces the survival of Capan-2 and BxPC-3 human pancreatic cancer cells in a dose-dependent manner. On the other hand, Triphala failed to cause any cytotoxic effects on the growth of HPDE-6 near normal pancreatic epithelial cells. Suppression of pancreatic cancer cell growth by Triphala in our model was due to induction of apoptosis, which in turn was associated with generation of ROS. Pretreatment of Capan-2 cells with antioxidant NAC blocked ROS generation and completely protected the cells from Triphala-induced apoptosis. Our results also demonstrate that Triphala treatment caused DNA damage resulting in the activation of ATM and ERK leading to stabilization of p53. Blocking ERK activation by MEK-1/2 inhibitor U0126 or p53 activation by pifithrin-α completely protected Capan-2 (wild type p53) cells from Triphala-induced apoptosis. Similarly, U0126 treatment blocked Triphala-induced apoptosis in BxPC-3 (mutated p53) cells, suggesting ERK as a molecular target of Triphala in pancreatic cancer cells. Further, orally feeding 50 mg/kg or 100 mg/kg Triphala to nude mice significantly retarded the growth of Capan-2 pancreatic tumor xenograft. Tumors from Triphala treated mice demonstrated increased apoptosis in the tumor cells, which was due to the activation of ERK and p53. To the best of our knowledge, this is the first study to report the molecular mechanism of the chemotherapeutic effects of Triphala against pancreatic cancer.
Reactive oxygen species (ROS) are the known mediators of intracellular signaling cascades. Excessive production of ROS nonetheless leads to oxidative stress, loss of cell function and apoptosis or necrosis [25
]. Our results reveal that Triphala-induced apoptosis in pancreatic cancer cells is initiated by ROS generation, the effect of which can be blocked by antioxidant NAC. Several previous studies including those from our laboratory have implicated ROS as a possible mechanism for DNA damage and induction of apoptosis [26
]. DNA damage plays a critical role in maintaining genomic integrity. Tumor cells exhibit genetic instability causing functional inactivation of p53 that plays an important role in DNA damage checkpoint pathways. In response to DNA damage, p53 is stabilized through phosphorylation at Ser 15 by ATM [22
]. The effects of Triphala are compatible with this assertion. Our results do indicate that Triphala treatment causes DNA damage as depicted by increased phosphorylation of H2A.X at Ser 139, an indicator for the presence of DNA double-strand breaks.
DNA damage has been shown to activate the kinase activity of ATM, which subsequently modifies a number of downstream targets including phosphorylation of p53 at Ser 15 at the N-terminus [33
]. Our studies reveal that Triphala treatment activates ATM by phosphorylation at Ser 1981. Moreover, our results also demonstrate increased protein expression and phosphorylation of p53 at Ser 15 in response to Triphala treatment. Stabilization of p53 by Triphala was further confirmed by nuclear transcriptional activity of p53. Induction of apoptosis by Triphala was almost completely blocked when the cells were pretreated with p53 specific inhibitor pifithrin, signifying the role of p53 in Triphala-induced apoptosis in pancreatic cancer cells.
A number of studies have shown the importance of ERK signaling pathway in regulating apoptosis [35
]. Although, ERK pathway delivers a survival signal, quite a few recent studies have linked the activation of ERK with induction of apoptosis by various chemopreventive and chemotherapeutic agents [39
]. In fact, oxidants have been shown to activate ERK by taking over the growth factor receptor signaling pathways [42
]. Moreover, ERK may get activated in response to DNA damage and can phosphorylate p53 in vitro
]. We found that exposure of Capan-2 or BxPC-3 cells with apoptosis-inducing concentration of Triphala results in a rapid and sustained activation of ERK in a concentration and time-dependent manner. Triphala mediated activation of ERK as well as apoptosis was completely abolished by MEK-1 inhibitor. MEK-1, which is an upstream of ERK, is also activated by Triphala in Capan-2 cells. Further, we observed that p53 is transcriptionaly regulated by ERK in response to Triphala treatment suggesting ERK as an upstream regulator of p53 in Capan-2 cells. We also observed that Triphala induce apoptosis by ERK activation in BxPC-3 cells, which has mutated p53. This is in part consistent with the observation that activated ERK lead to apoptosis after DNA damage in a p53 independent manner [49
]. On the other hand, Triphala is not at all toxic to HPDE-6 normal pancreatic epithelial cells and does not activate ERK, p53 or caspases. Taken together, our results indicate ERK as a possible molecular target of Triphala in pancreatic cancer cells.
Pancreatic tumor growth inhibition and induction of apoptosis in vivo
was observed by the oral administration of 50 mg/kg or 100 mg/kg Triphala 5 times a week. Our results are consistent with previous studies where Triphala was shown to be effective in suppressing the growth of Barc-95 (mouse thymic lymphoma) xenograft in mice [15
]. Although, pharmacokinetics of Triphala in humans has not been determined, it has been used safely for centuries in the Ayuervedic medicinal system in India for the treatment of various gastrointestinal-related disorders. The effective dose of Triphala in our animal model for suppressing tumor growth, if extrapolated to humans ranges from 4 to 8 grams per day for a person weighing 70 kg. These doses of Triphala come within the dose range already being used by humans in countries such as India. The overall incidence of pancreatic cancer is approximately 8–10 cases per 100,000 persons per year in the USA [50
]. In other countries the incidence may vary from 8–12 cases per 100,000 persons per year [51
]. However, in some areas of the world, pancreatic cancer is sporadic; for instance, the incidence of pancreatic cancer in India is less than 2 cases per 100,000 persons per year [51
]. It is tempting to speculate that the low incidence of pancreatic cancer in India may in part be due to consumption of Triphala or one of its constituent Amla (rich in ascorbic acid). Nevertheless, detailed epidemiological studies are required to substantiate this assumption. Interestingly, a very recent study demonstrated the anti-tumoral effects of ascorbic acid (component of Triphala) against ovarian, pancreatic and glioblastoma xenografts in mice [54
]. Our recent studies have observed that Amla is also very effective against pancreatic cancer (manuscript communicated).