Triptolide Induces Cell Death in Pancreatic Cancer Cells by Apoptotic and Autophagic Pathways
ABSTRACT 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.
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ABSTRACT: Pancreatic cancer has a 5-year survival rate of less than 5%, partly because of limited chemotherapeutic options, thereby highlighting the need for novel therapies. Triptolide, a diterpene triepoxide that was derived from a Chinese herb, has shown great promise in preclinical testing against pancreatic cancer using immunocompromised animals. In this study, we tested the ability of triptolide to induce cell death in cell lines derived from a primary tumor and adjacent liver metastases of immunocompetent animals (Kras, Trp53, Pdx-1 Cre [KPC]). Both cell lines were more aggressive in their ability to form tumors when compared with other pancreatic cancer cell lines and showed constitutive activation of the nuclear factor κ-light-chain-enhancer of activated B cells pathway. Triptolide induced apoptotic cell death in both cell lines, as evidenced by decreased cell viability as well as increased caspase 3/7 activity, annexin V positivity, and increased terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling positivity in tumors from KPC animals treated with Minnelide. In addition, triptolide decreased levels of HSP70, its transcription factor HSF1, as well as the antiapoptotic proteins Bcl-xL, Bcl-2, and Mcl-1, which are known to be up-regulated in pancreatic cancer. The ability of triptolide to cause cell death in cell lines derived from immunocompetent animals further validates its potential as a novel agent against pancreatic cancer.Pancreas 05/2015; 44(4):583-9. DOI:10.1097/MPA.0000000000000317 · 3.01 Impact Factor
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ABSTRACT: Epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs), including gefitinib, are effective for non-small cell lung cancer (NSCLC) patients with EGFR mutations. However, these patients eventually develop resistance to EGFR-TKI. The goal of the present study was to investigate the involvement of autophagy in gefitinib resistance. We developed gefitinib-resistant cells (PC-9/gef) from PC-9 cells (containing exon 19 deletion EGFR) after long-term exposure in gefitinib. PC-9/gef cells (B4 and E3) were 200-fold more resistant to gefitinib than PC-9/wt cells. Compared with PC-9/wt cells, both PC-9/gefB4 and PC-9/gefE3 cells demonstrated higher basal LC3-II levels which were inhibited by 3-methyladenine (3-MA, an autophagy inhibitor) and potentiated by chloroquine (CQ, an inhibitor of autophagolysosomes formation), indicating elevated autophagy in PC-9/gef cells. 3-MA and CQ concentration-dependently inhibited cell survival of both PC-9wt and PC-9/gef cells, suggesting that autophagy may be pro-survival. Furthermore, gefitinib increased LC3-II levels and autolysosome formation in both PC-9/wt cells and PC-9/gef cells. In PC-9/wt cells, CQ potentiated the cytotoxicity by low gefitinib (3nM). Moreover, CQ overcame the acquired gefitinib resistance in PC-9/gef cells by enhancing gefitinib-induced cytotoxicity, activation of caspase 3 and poly (ADP-ribose) polymerase cleavage. Using an in vivo model xenografting with PC-9/wt and PC-9/gefB4 cells, oral administration of gefitinib (50 mg/kg) completely inhibited the tumor growth of PC-9/wt but not PC-9/gefB4cells. Combination of CQ (75 mg/kg, i.p.) and gefitinib was more effective than gefitinib alone in reducing the tumor growth of PC-9/gefB4. Our data suggest that inhibition of autophagy may be a therapeutic strategy to overcome acquired resistance of gefitinib in EGFR mutation NSCLC patients.PLoS ONE 01/2015; 10(3):e0119135. DOI:10.1371/journal.pone.0119135 · 3.53 Impact Factor
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ABSTRACT: Autophagy is a complicated self-eating response of cells to external or internal stimuli. This process involves cellular degradation through the lysosomes of dysfunctional or unnecessary cellular components or organelles to maintain basic energy levels. This nonapoptotic programmed cell death, similar to other main phenomena of cell biology such as apoptosis and differentiation, has been implicated in the pathogenesis of a series of disorders, such as neurodegenerative diseases, cardiovascular diseases, and especially in cancer. Increasing evidence has suggested that the autophagy pathways may provide potential targets for cancer intervention, although their precise roles in cancer initiation and progression remain controversial. Natural products are very important sources of chemotherapeutics agents. Regulation of autophagy could be an important mechanism contributing to the beneficial effect of quite a few natural products. Herein, we briefly introduce the characteristics and roles of autophagy in cancer and systematically summarize the natural autophagy regulators, with emphasis on apigenin, berberine, beta-elemene, capsaicin, curcumin, genistein, kaempferol, oridonin, paclitaxel, quercetin, resveratrol, silybin, triptolide, and ursolic acid, with the aim to provide information for novel avenues on cancer therapies based on autophagy.Phytochemistry Reviews 02/2014; 14(1). DOI:10.1007/s11101-014-9339-3 · 2.89 Impact Factor