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

<p>Abstract</p> <p>Background</p> <p>Triphala is commonly used in Ayurvedic medicine to treat variety of diseases; however its mechanism of action remains unexplored. This study elucidates the molecular mechanism of Triphala against human pancreatic cancer in the cellular and in vivo model.</p> <p>Methods</p> <p>Growth-inhibitory effects of Triphala were evaluated in Capan-2, BxPC-3 and HPDE-6 cells by Sulphoradamine-B assay. Apoptosis was determined by cell death assay and western blotting. Triphala was administered orally to nude mice implanted with Capan-2 xenograft. Tumors were analyzed by immunohistochemistry and western blotting.</p> <p>Results</p> <p>Exposure of Capan-2 cells to the aqueous extract of Triphala for 24 h resulted in the significant decrease in the survival of cells in a dose-dependent manner with an IC50 of about 50 μg/ml. Triphala-mediated reduced cell survival correlated with induction of apoptosis, which was associated with reactive oxygen species (ROS) generation. Triphala-induced apoptosis was linked with phosphorylation of p53 at Ser-15 and ERK at Thr-202/Tyr-204 in Capan-2 cells. Above mentioned effects were significantly blocked when the cells were pretreated with an antioxidant N-acetylcysteine (NAC), suggesting the involvement of ROS generation. Pretreatment of cells with pifithrin-α or U0126, specific inhibitors of p53 or MEK-1/2, significantly attenuated Triphala-induced apoptosis. Moreover, NAC or U0126 pretreatment significantly attenuated Triphala-induced p53 transcriptional activity. Similarly, Triphala induced apoptosis in another pancreatic cancer cell line BxPC-3 by activating ERK. On the other hand, Triphala failed to induce apoptosis or activate ERK or p53 in normal human pancreatic ductal epithelial (HPDE-6) cells. Further, oral administration of 50 mg/kg or 100 mg/kg Triphala in PBS, 5 days/week significantly suppressed the growth of Capan-2 pancreatic tumor-xenograft. Reduced tumor-growth in Triphala fed mice was due to increased apoptosis in the tumors cells, which was associated with increased activation of p53 and ERK.</p> <p>Conclusion</p> <p>Our preclinical studies demonstrate that Triphala is effective in inhibiting the growth of human pancreatic cancer cells in both cellular and in vivo model. Our data also suggests that the growth inhibitory effects of Triphala is mediated by the activation of ERK and p53 and shows potential for the treatment and/or prevention of human pancreatic cancer.</p

Systemic Analysis of Heat Shock Response Induced by Heat Shock and a Proteasome Inhibitor MG132

The molecular basis of heat shock response (HSR), a cellular defense mechanism against various stresses, is not well understood. In this, the first comprehensive analysis of gene expression changes in response to heat shock and MG132 (a proteasome inhibitor), both of which are known to induce heat shock proteins (Hsps), we compared the responses of normal mouse fibrosarcoma cell line, RIF- 1, and its thermotolerant variant cell line, TR-RIF-1 (TR), to the two stresses. The cellular responses we examined included Hsp expressions, cell viability, total protein synthesis patterns, and accumulation of poly-ubiquitinated proteins. We also compared the mRNA expression profiles and kinetics, in the two cell lines exposed to the two stresses, using microarray analysis. In contrast to RIF-1 cells, TR cells resist heat shock caused changes in cell viability and whole-cell protein synthesis. The patterns of total cellular protein synthesis and accumulation of poly-ubiquitinated proteins in the two cell lines were distinct, depending on the stress and the cell line. Microarray analysis revealed that the gene expression pattern of TR cells was faster and more transient than that of RIF-1 cells, in response to heat shock, while both RIF-1 and TR cells showed similar kinetics of mRNA expression in response to MG132. We also found that 2,208 genes were up-regulated more than 2 fold and could sort them into three groups: 1) genes regulated by both heat shock and MG132, (e.g. chaperones); 2) those regulated only by heat shock (e.g. DNA binding proteins including histones); and 3) those regulated only by MG132 (e.g. innate immunity and defense related molecules). This study shows that heat shock and MG132 share some aspects of HSR signaling pathway, at the same time, inducing distinct stress response signaling pathways, triggered by distinct abnormal proteins