Pancreatic adenocarcinoma, among the most lethal human malignancies, is resistant to current chemotherapies. We previously showed that triptolide inhibits the growth of pancreatic cancer cells in vitro and prevents tumor growth in vivo. This study investigates the mechanism by which triptolide kills pancreatic cancer cells.
Cells were treated with triptolide and viability and caspase-3 activity were measured using colorimetric assays. Annexin V, propidium iodide, and acridine orange staining were measured by flow cytometry. Immunofluorescence was used to monitor the localization of cytochrome c and Light Chain 3 (LC3) proteins. Caspase-3, Atg5, and Beclin1 levels were down-regulated by exposing cells to their respective short interfering RNA.
We show that triptolide induces apoptosis in MiaPaCa-2, Capan-1, and BxPC-3 cells and induces autophagy in S2-013, S2-VP10, and Hs766T cells. Triptolide-induced autophagy has a pro-death effect, requires autophagy-specific genes, atg5 or beclin1, and is associated with the inactivation of the Protein kinase B (Akt)/mammalian target of Rapamycin/p70S6K pathway and the up-regulation of the Extracellular Signal-Related Kinase (ERK)1/2 pathway. Inhibition of autophagy in S2-013 and S2-VP10 cells results in cell death via the apoptotic pathway whereas inhibition of both autophagy and apoptosis rescues cell death.
This study shows that triptolide kills pancreatic cancer cells by 2 different pathways. It induces caspase-dependent apoptotic death in MiaPaCa-2, Capan-1, and BxPC-3, and induces caspase-independent autophagic death in metastatic cell lines S2-013, S2-VP10, and Hs766T, thereby making it an attractive chemotherapeutic agent against a broad spectrum of pancreatic cancers.
"Autophagosome nucleation is initiated by the PI3 kinase type III-Atg6/Beclin 1 complex, while the elongation is monitored by Atg12-Atg5 and Atg8/LC3-phosphatidylethanolamine conjugate systems, both of which are key characteristics of autophagy , , . Autophagy can be induced by a number of chemotherapeutic agents such as arsenic trioxide and oxaliplatin , : however, the role of autophagy in cancer is controversial , , . A regulated autophagic response can ensure the physiological turnover of damaged organelles and recycled macromolecules to meet the energy demands in response to cytotoxic drugs, leading to prolonged cell survival , . "
[Show abstract][Hide abstract] ABSTRACT: Gambogic acid (GA), the main active component of gamboge resin, has potent antitumor activity both in vivo and in vitro. However, the underlying molecular mechanisms remain unclear. In this study, we found that GA could initiate autophagy in colorectal cancer cells, and inhibition of the autophagy process accelerated the effect of proliferative inhibition and apoptotic cell death induced by GA, implying a protective role of autophagy. Two-dimensional electrophoresis-based proteomics showed that GA treatment altered the expression of multiple proteins involved in redox signaling and lipid metabolism. Functional studies revealed that GA-induced dysregulation of lipid metabolism could activate 5-lipoxygenase (5-LOX), resulting in intracellular ROS accumulation, followed by inhibition of Akt-mTOR signaling and autophagy initiation. Finally, results using a xenograft model suggested ROS-induced autophagy protect against the antitumor effect of GA. Taken together, these data showed new biological activities of GA against colorectal cancer underlying the protective role of ROS-induced autophagy. This study will provide valuable insights for future studies regarding the anticancer mechanisms of GA.
PLoS ONE 05/2014; 9(5):e96418. DOI:10.1371/journal.pone.0096418 · 3.23 Impact Factor
"As the major active component in leigongteng, triptolide has attracted high attention for its multiple activities, especially for its anti-tumor effect . Triptolide has shown a broad-spectrum of anti-tumor effect and cytotoxicity on almost all kinds of cancers, including lung cancer , pancreatic cancer , breast cancer , ovary cancer , renal cancer , glioblastoma , thyroid cancer , leukemia , colon cancer . Our previous study also found that triptolide shows effective anti-PCa activity . "
[Show abstract][Hide abstract] ABSTRACT: Triptolide, an active compound extracted from Chinese herb Leigongteng (Tripterygium wilfordii Hook F.), shows a broad-spectrum of anticancer activity through its cytotoxicity. However, the efficacy of triptolide on laryngocarcinoma rarely been evaluated, and the mechanism by which triptolide-induced cellular apoptosis is still not well understood. In this study, we found that triptolide significantly inhibited the laryngocarcinoma HEp-2 cells proliferation, migration and survivability. Triptolide induces HEp-2 cell cycle arrest at the G1 phase and apoptosis through intrinsic and extrinsic pathways since both caspase-8 and -9 are activated. Moreover, triptolide enhances p53 expression by increasing its stability via down-regulation of E6 and E6AP. Increased p53 transactivates down-stream target genes to initiate apoptosis. In addition, we found that short time treatment with triptolide induced DNA damage, which was consistent with the increase in p53. Furthermore, the cytotoxicity of triptolide is decreased by p53 knockdown or use of caspases inhibitor. In conclusion, our results demonstrated that triptolide inhibits cell proliferation and induces apoptosis in laryngocarcinoma cells by enhancing p53 expression and activating p53 functions through induction of DNA damage and suppression of E6 mediated p53 degradation. These studies indicate that triptolide is a potential anti-laryngocarcinoma drug.
PLoS ONE 11/2013; 8(11):e80784. DOI:10.1371/journal.pone.0080784 · 3.23 Impact Factor
"We and others have identified triptolide, a diterpene triepoxide derived from a Chinese plant, Tripterygium wilfordii, as a potential chemotherapeutic agent against pancreatic, breast and colon cancers, as well as cholangiocarcinoma, osteosarcoma and neuroblastoma
[17-20]. Our group has shown that triptolide is capable of inducing apoptotic as well as autophagy as a mechanism of cell death in some pancreatic cancer cell lines
. Although triptolide is shown to be a very effective compound in vitro, its use in clinical settings is limited owing to its low solubility. "
[Show abstract][Hide abstract] ABSTRACT: Pancreatic cancer is one of the most lethal human malignancies, with an all-stage 5-year survival of <5%, mainly due to lack of effective available therapies. Cancer cell survival is dependent upon up-regulation of the pro-survival response, mediated by anti-apoptotic proteins such as Mcl-1.
Here we show that over-expression of Mcl-1 in pancreatic patient tumor samples are linked to advancement of the disease. We have previously shown that triptolide, a diterpene triepoxide, is effective both in vitro and in vivo, in killing pancreatic cancer cells. Decrease of Mcl-1 levels, either by siRNA or by treatment with triptolide results in cell death. Using pancreatic cancer cell lines, we have shown that miR-204, a putative regulator of Mcl-1, is repressed in cancer cell lines compared to normal cells. Over-expression of miR-204, either by a miR-204 mimic, or by triptolide treatment results in a decrease in Mcl-1 levels, and a subsequent decrease in cell viability. Using luciferase reporter assays, we confirmed the ability of miR-204 to down-regulate Mcl-1 by directly binding to the Mcl-1 3' UTR. Using human xenograft samples treated with Minnelide, a water soluble variant of triptolide, we have shown that miR-204 is up-regulated and Mcl-1 is down-regulated in treated vs. control tumors.
Triptolide mediated miR-204 increase causes pancreatic cancer cell death via loss of Mcl-1.
Molecular Cancer 09/2013; 12(1):105. DOI:10.1186/1476-4598-12-105 · 4.26 Impact Factor
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