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

Activation of the steroid and xenobiotic receptor, SXR, induces apoptosis in breast cancer cells

<p>Abstract</p> <p>Background</p> <p>The steroid and xenobiotic receptor, SXR, is an orphan nuclear receptor that regulates metabolism of diverse dietary, endobiotic, and xenobiotic compounds. SXR is expressed at high levels in the liver and intestine, and at lower levels in breast and other tissues where its function was unknown. Since many breast cancer preventive and therapeutic compounds are SXR activators, we hypothesized that some beneficial effects of these compounds are mediated through SXR.</p> <p>Methods</p> <p>To test this hypothesis, we measured proliferation of breast cancer cells in response to SXR activators and evaluated consequent changes in the expression of genes critical for proliferation and cell-cycle control using quantitative RT-PCR and western blotting. Results were confirmed using siRNA-mediated gene knockdown. Statistical analysis was by t-test or ANOVA and a P value ≤ 0.05 was considered to be significant.</p> <p>Results</p> <p>Many structurally and functionally distinct SXR activators inhibited the proliferation of MCF-7 and ZR-75-1 breast cancer cells by inducing cell cycle arrest at the G1/S phase followed by apoptosis. Decreased growth in response to SXR activation was associated with stabilization of p53 and up-regulation of cell cycle regulatory and pro-apoptotic genes such as p21, PUMA and BAX. These gene expression changes were preceded by an increase in inducible nitric oxide synthase and nitric oxide in these cells. Inhibition of iNOS blocked the induction of p53. p53 knockdown inhibited up-regulation of p21 and BAX. We infer that NO is required for p53 induction and that p53 is required for up-regulation of cell cycle regulatory and apoptotic genes in this system. SXR activator-induced increases in iNOS levels were inhibited by siRNA-mediated knockdown of SXR, indicating that SXR activation is necessary for subsequent regulation of iNOS expression.</p> <p>Conclusion</p> <p>We conclude that activation of SXR is anti-proliferative in p53 wild type breast cancer cells and that this effect is mechanistically dependent upon the local production of NO and NO-dependent up-regulation of p53. These findings reveal a novel biological function for SXR and suggest that a subset of SXR activators may function as effective therapeutic and chemo-preventative agents for certain types of breast cancers.</p

Synthesis of a Dual Functional Anti-MDR Tumor Agent PH II-7 with Elucidations of Anti-Tumor Effects and Mechanisms

Multidrug resistance mediated by P-glycoprotein in cancer cells has been a major issue that cripples the efficacy of chemotherapy agents. Aimed for improved efficacy against resistant cancer cells, we designed and synthesized 25 oxindole derivatives based on indirubin by structure-activity relationship analysis. The most potent one was named PH II-7, which was effective against 18 cancer cell lines and 5 resistant cell lines in MTT assay. It also significantly inhibited the resistant xenograft tumor growth in mouse model. In cell cycle assay and apoptosis assay conducted with flow cytometry, PH II-7 induced S phase cell cycle arrest and apoptosis even in resistant cells. Consistently revealed by real-time PCR, it modulates the expression of genes related to the cell cycle and apoptosis in these cells, which may contributes to its efficacy against them. By side-chain modification and FITC-labeling of PH II-7, we were able to show with confocal microscopy that not only it was not pumped by P-glycoprotein, it also attenuated the efflux of Adriamycin by P-glycoprotein in MDR tumor cells. Real-time PCR and western blot analysis showed that PH II-7 down-regulated MDR1 gene via protein kinase C alpha (PKCA) pathway, with c-FOS and c-JUN as possible mediators. Taken together, PH II-7 is a dual-functional compound that features both the cytotoxicity against cancer cells and the inhibitory effect on P-gp mediated drug efflux

p53-independent apoptosis induced by muscle differentiation stimuli in polyomavirus large T-expressing myoblasts

Abnormal proliferation signals, driven by cellular or viral oncogenes, can result in the induction of apoptosis under sub-optimal cell growth conditions. The tumor suppressor p53 plays a central role in mediating oncogene-induced apoptosis, therefore transformed cells lacking p53 are generally resistant to apoptosis-promoting treatments. In a previous work we have reported that the expression of polyomavirus large T antigen causes apoptosis in differentiating myoblasts and that this phenomenon is dependent on the onset of muscle differentiation in the absence of a correct cell cycle arrest. Here we report that polyomavirus large T increases the levels and activity of p53, but these alterations are not involved in the apoptotic mechanism. Apoptosis in polyomavirus large T-expressing myoblasts is not prevented by the expression of a p53 dominant-negative mutant nor it is increased by p53 overexpression. Moreover, forced differentiation induced through the over-expression of the muscle regulatory factor MyoD, leads to apoptosis without altering p53 function and, more significantly, even in a p53-null background. Our results indicate that apoptosis induced by the activation of muscle differentiation pathways in oncogene-expressing cells can occur in a p53-independent manner

Double-stranded internucleosomal cleavage of apoptotic DNA is dependent on the degree of differentiation in muscle cells

Apoptotic cell death has been correlated to DNA fragmentation into discrete segments corresponding to the length of nucleosomal protected fragments of 180-200 base pairs or multiples of it, This DNA degradation has been ascribed to endonuclease activity that cleaves internucleosomally, thus giving rise to a ladder distribution upon electrophoretic migration. This strict correlation was, however, shown to have notable exceptions, since in some cases only single strand cleavage in the internucleosomal DNA regions has been observed (Tomei, D. L,, Shapiro, P, J,, and Cope, O. F. (1993) Proc, Natl. Acad. Sci, U. S. A. 90, 853-857), In the present work we show that mouse muscle cells, able to differentiate in vitro, if subjected to apoptosis present no DNA degradation into ladder form unless differentiation is previously induced. Furthermore, C3H/10T1/2 fibroblast cells, known to undergo apoptosis without DNA ladder formation, if converted to a myogenic program by MyoD expression, display internucleosomal DNA degradation upon induction of differentiation

Multiple biochemical properties of the p53 molecule contribute to activation of polymerase iota-dependent DNA damage tolerance

Abstract We have previously reported that p53 decelerates nascent DNA elongation in complex with the translesion synthesis (TLS) polymerase ι (POLι) which triggers a homology-directed DNA damage tolerance (DDT) pathway to bypass obstacles during DNA replication. Here, we demonstrate that this DDT pathway relies on multiple p53 activities, which can be disrupted by TP53 mutations including those frequently found in cancer tissues. We show that the p53-mediated DDT pathway depends on its oligomerization domain (OD), while its regulatory C-terminus is not involved. Mutation of residues S315 and D48/D49, which abrogate p53 interactions with the DNA repair and replication proteins topoisomerase I and RPA, respectively, and residues L22/W23, which disrupt formation of p53-POLι complexes, all prevent this DDT pathway. Our results demonstrate that the p53-mediated DDT requires the formation of a DNA binding-proficient p53 tetramer, recruitment of such tetramer to RPA-coated forks and p53 complex formation with POLι. Importantly, our mutational analysis demonstrates that transcriptional transactivation is dispensable for the POLι-mediated DDT pathway, which we show protects against DNA replication damage from endogenous and exogenous sources