Background & Aims Pancreatic adenocarcinoma, among the most lethal human malignancies, is resistant to current chemotherapies. We have previously shown 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, which has not been previously studied. Methods 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 LC3 proteins. Caspase-3, Atg5 and Beclin1 levels were downregulated by exposing cells to their respective siRNA. Results We show that triptolide induces apoptosis in MiaPaCa-2, Capan-1 and BxPC-3 cells and 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 Akt/mTOR/p70S6K pathway and the upregulation of the ERK1/2 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. Conclusions This study shows, for the first time, that triptolide kills pancreatic cancer cells by two different pathways. It induces caspase-dependent apoptotic death in MiaPaCa-2, Capan-1 and BxPC-3 and 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.

Pancreatic ductal adenocarcinoma (PDAC), one of the deadliest malignancies, is resistant to current chemotherapies. We previously showed that triptolide inhibits PDAC cell growth in vitro and blocks metastatic spread in vivo. Triptolide downregulates heat shock protein 70 (HSP70), a molecular chaperone upregulated in several tumor types. This study investigates the mechanism by which triptolide inhibits HSP70. As microRNAs (miRNAs) are becoming increasingly recognized as negative regulators of gene expression, we tested whether triptolide regulates HSP70 via miRNAs. Here we show that triptolide, as well as quercetin but not gemcitabine, upregulated miR-142-3p in PDAC cells (MIA PaCa-2, Capan-1, and S2-013). Ectopic expression of miR-142-3p inhibited cell proliferation, measured by Electric Cell-substrate Impedance Sensing, and decreased HSP70 expression, measured by real-time PCR and immunoblotting, compared with controls. We demonstrated that miR-142-3p directly binds to the 3’UTR of HSP70, and that this interaction is important as HSP70 overexpression rescued miR-142-3p-induced cell death. We found that miR-142-3p regulates HSP70 independently of heat shock factor 1. Furthermore, Minnelide, a water soluble prodrug of triptolide, induced the expression of miR-142-3p in vivo. This is the first description of an miRNA-mediated mechanism of HSP70 regulation in cancer, making miR-142-3p an attractive target for PDAC therapeutic intervention.

Osteosarcoma is the most common bone cancer in children and adolescents with a five-year survival rate of about 70%. In this study, we have evaluated the preclinical therapeutic efficacy of the novel synthetic drug, Minnelide, a prodrug of triptolide on osteosarcoma. Triptolide was effective in significantly inducing apoptosis in all osteosarcoma cell lines tested but had no significant effect on the human osteoblast cells. Notably, Minnelide treatment significantly reduced tumor burden and lung metastasis in the orthotopic and lung colonization models. Triptolide/Minnelide effectively downregulated the levels of pro-survival proteins such as heat shock proteins, cMYC, survivin and targets NF-κB pathway.

BackgroundPancreatic 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.ResultsHere we show that over-expression of Mcl-1 in pancreatic patient tumor samples is 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.ConclusionTriptolide mediated miR-204 increase causes pancreatic cancer cell death via loss of Mcl-1.

BackgroundMUC1 is a type I transmembrane glycoprotein aberrantly overexpressed in various cancer cells including pancreatic cancer. The cytosolic end of MUC1 (MUC1-c) is extensively involved in a number of signaling pathways. MUC1-c is reported to inhibit apoptosis in a number of cancer cells, but the mechanism of inhibition is unclear.MethodExpression of MUC1-c was studied in the pancreatic cancer cell line MIAPaCa-2 at the RNA level by using qRTPCR and at the protein level by Western blotting. MUC1-c expression was inhibited either by siRNA or by a specific peptide inhibitor, GO-201. Effect of MUC1-c inhibition on viability and proliferation and lysosomal permeabilization were studied. Association of MUC1-c with HSP70 was detected by co-immunoprecipitation of MUC1-c and HSP70. Localization of MUC1-c in cellular organelles was monitored by immunofluorescence and with immuno- blotting by MUC1-c antibody after subcellular fractionation.ResultsInhibition of MUC1-c by an inhibitor (GO-201) or siRNA resulted in reduced viability and reduced proliferation of pancreatic cancer cells. Furthermore, GO-201, the peptide inhibitor of MUC1-c, was effective in reducing tumor burden in pancreatic cancer mouse model. MUC1-c was also found to be associated with HSP70 in the cytosol, although a significant amount of MUC1 was also seen to be present in the lysosomes. Inhibition of MUC1 expression or activity showed an enhanced Cathepsin B activity in the cytosol, indicating lysosomal permeabilization. Therefore this study indicates that MUC1-c interacted with HSP70 in the cytosol of pancreatic cancer cells and localized to the lysosomes in these cells. Further, our results showed that MUC1-c protects pancreatic cancer cells from cell death by stabilizing lysosomes and preventing release of Cathepsin B in the cytosol.