piplartine and Pancreatic-Neoplasms

Piperlongumine (PL) is an alkaloid that inhibits glutathione S-transferase pi 1 (GSTP1) activity, resulting in elevated reactive oxygen species (ROS) levels and cancer-selective cell death. We aimed to identify stress-associated molecular responses to PL treatment in pancreatic ductal adenocarcinoma (PDAC) cells. GSTP1 directly interacts with JNK, which is activated by oxidative stress and can lead to decreased cancer cell proliferation and cell death. Therefore, we hypothesized that JNK pathways are activated in response to PL treatment. Our results show PL causes dissociation of GSTP1 from JNK; robust JNK, c-Jun, and early ERK activation followed by suppression; increased expression of cleaved caspase-3 and cleaved PARP; and nuclear translocation of Nrf2 and c-Myc in PDAC cells. Gene expression analysis revealed PL caused a > 20-fold induction of heme oxygenase-1 (HO-1), which we hypothesized was a survival mechanism for PDAC cells under enhanced oxidative stress. HO-1 knockout resulted in enhanced PL-induced PDAC cell death under hypoxic conditions. Similarly, high concentrations of the HO-1 inhibitor, ZnPP (10 µM), sensitized PDAC cells to PL; however, lower concentrations ZnPP (10 nM) and high or low concentrations of SnPP both protected PDAC cells from PL-induced cell death. Interestingly, the JNK inhibitor significantly blocked PL-induced PDAC cell death, Nrf-2 nuclear translocation, and HMOX-1 mRNA expression. Collectively, the results demonstrate JNK signaling contributes to PL-induced PDAC cell death, and at the same time, activates Nrf-2 transcription of HMOX-1 as a compensatory survival mechanism. These results suggest that elevating oxidative stress (using PL) while at the same time impairing antioxidant capacity (inhibiting HO-1) may be an effective therapeutic approach for PDAC.

Topics: Alkaloids; Apoptosis; Cell Line, Tumor; Dioxolanes; Heme Oxygenase-1; Humans; JNK Mitogen-Activated Protein Kinases; MAP Kinase Signaling System; NF-E2-Related Factor 2; Oxidative Stress; Pancreatic Neoplasms; Reactive Oxygen Species; Transcriptional Activation; Transcriptome

Pancreatic cancer is one of the most lethal types of cancer with a mortality rate of almost 95%. Treatment with current chemotherapeutic drugs has limited success due to poor responses. Therefore, the development of novel drugs or effective combination therapies is urgently required. Piperlongumine (PL) is a natural product with cytotoxic properties restricted to cancer cells by significantly increasing intracellular reactive oxygen species (ROS) levels. In the present study, we demonstrated that PL induced cancer cell death through, at least in part, the induction of ferroptosis, as the cancer cell-killing activity was inhibited by the antioxidant, N‑acetylcysteine, ferroptosis inhibitors (ferrostatin‑1 and liproxstatin‑1) and the iron chelator, deferoxamine (DFO), but not by the apoptosis inhibitor, Z-VAD-FMK, or the necrosis inhibitor, necrostatin‑1. Cotylenin A (CN‑A; a plant growth regulator) exhibits potent antitumor activities in several cancer cell lines, including pancreatic cancer cell lines. We found that CN‑A and PL synergistically induced the death of pancreatic cancer MIAPaCa‑2 and PANC‑1 cells for 16 h. CN‑A enhanced the induction of ROS by PL for 4 h. The synergistic induction of cell death was also abrogated by the ferroptosis inhibitors and DFO. The present results revealed that clinically approved sulfasalazine (SSZ), a ferroptosis inducer, enhanced the death of pancreatic cancer cells induced by PL and the combined effects were abrogated by the ferroptosis inhibitors and DFO. SSZ further enhanced the cancer cell-killing activities induced by combined treatment with PL plus CN‑A. On the other hand, the synergistic induction of cell death by PL and CN‑A was not observed in mouse embryonic fibroblasts (MEFs), and SSZ did not enhance the death of MEFs induced by PL plus CN‑A. These results suggest that the triple combined treatment with PL, CN‑A and SSZ is highly effective against pancreatic cancer.

Topics: Acetylcysteine; Amino Acid Chloromethyl Ketones; Animals; Antineoplastic Combined Chemotherapy Protocols; Cell Death; Cell Line, Tumor; Cyclohexylamines; Deferoxamine; Dioxolanes; Diterpenes; Drug Synergism; Fibroblasts; Humans; Iron; Mice; Pancreatic Neoplasms; Phenylenediamines; Quinoxalines; Reactive Oxygen Species; Spiro Compounds; Sulfasalazine

Glutathione S-transferase pi 1 (GSTP1) is frequently overexpressed in cancerous tumors and is a putative target of the plant compound piperlongumine (PL), which contains two reactive olefins and inhibits proliferation in cancer cells but not normal cells. PL exposure of cancer cells results in increased reactive oxygen species and decreased GSH. These data in tandem with other information led to the conclusion that PL inhibits GSTP1, which forms covalent bonds between GSH and various electrophilic compounds, through covalent adduct formation at the C7-C8 olefin of PL, whereas the C2-C3 olefin of PL was postulated to react with GSH. However, direct evidence for this mechanism has been lacking. To investigate, we solved the X-ray crystal structure of GSTP1 bound to PL and GSH at 1.1 Å resolution to rationalize previously reported structure activity relationship studies. Surprisingly, the structure showed that a hydrolysis product of PL (hPL) was conjugated to glutathione at the C7-C8 olefin, and this complex was bound to the active site of GSTP1; no covalent bond formation between hPL and GSTP1 was observed. Mass spectrometry (MS) analysis of the reactions between PL and GSTP1 confirmed that PL does not label GSTP1. Moreover, MS data also indicated that nucleophilic attack on PL at the C2-C3 olefin led to PL hydrolysis. Although hPL inhibits GSTP1 enzymatic activity in vitro, treatment of cells susceptible to PL with hPL did not have significant anti-proliferative effects, suggesting that hPL is not membrane-permeable. Altogether, our data suggest a model wherein PL is a prodrug whose intracellular hydrolysis initiates the formation of the hPL-GSH conjugate, which blocks the active site of and inhibits GSTP1 and thereby cancer cell proliferation.

Topics: Cell Proliferation; Crystallography, X-Ray; Dioxolanes; Glutathione; Glutathione S-Transferase pi; Humans; Mass Spectrometry; Pancreatic Neoplasms; Protein Binding; Protein Conformation; Tumor Cells, Cultured

Pancreatic cancer is an aggressive malignancy, which generally respond poorly to chemotherapy. Hence, novel agents that are safe and effective are highly needed. The aim of this study was to investigate whether piperlongumine, a natural product isolated from the fruit of the pepper Piper longum, has any efficacy against human pancreatic cancer when used either alone or in combination with gemcitabine in vitro and in a xenograft mouse model. In vitro, piperlongumine inhibited the proliferation of pancreatic cancer cell lines, potentiated the apoptotic effects of gemcitabine, inhibited the constitutive and inducible activation of NF-κB, and suppressed the NF-κB-regulated expression of c-Myc, cyclin D1, Bcl-2, Bcl-xL, Survivin, XIAP, VEGF, and matrix metalloproteinase-9 (MMP-9). Furthermore, in an in vivo xenograft model, we found piperlongumine alone significantly suppressed tumor growth and enhanced the antitumor properties of gemcitabine. These results were consistent with the downregulation of NF-κB activity and its target genes, decreased proliferation (PCNA and Ki-67), decreased microvessel density (CD31), and increased apoptosis (TUNEL) in tumor remnants. Collectively, our results suggest that piperlongumine alone exhibits significant antitumor effects against human pancreatic cancer and it further enhances the therapeutic effects of gemcitabine, possibly through the modulation of NF-κB- and NF-κB-regulated gene products.

Topics: Animals; Antimetabolites, Antineoplastic; Apoptosis; Blotting, Western; Cell Proliferation; Deoxycytidine; Dioxolanes; Drug Synergism; Drug Therapy, Combination; Gemcitabine; Gene Expression Regulation, Neoplastic; Humans; Immunoenzyme Techniques; Male; Mice; Mice, Inbred BALB C; Mice, Nude; NF-kappa B; Pancreatic Neoplasms; Signal Transduction; Tumor Cells, Cultured; Xenograft Model Antitumor Assays

Piperlongumine (PL), an alkaloid obtained from long peppers, displays antitumorigenic properties for a variety of human cell- and animal-based models. The aim of this study was to identify the underlying molecular mechanisms for PL anticancer effects on human pancreatic cancer cells. RNA sequencing (RNA-seq) was used to identify the effects of PL on the transcriptome of MIA PaCa-2 human pancreatic cancer cells. PL treatment of pancreatic cancer cells resulted in differential expression of 683 mRNA transcripts with known protein functions, 351 of which were upregulated and 332 of which were downregulated compared to control-treated cells. Transcripts associated with oxidative stress, endoplasmic reticulum (ER) stress, and unfolded protein response pathways were significantly overexpressed with PL treatment. Reverse transcription-quantitative polymerase chain reaction and western blotting were used to validate the RNA-seq results, which included upregulation of HO-1, IRE1α, cytochrome c, and ASNS. The results provide key insight into the mechanisms by which PL alters cancer cell physiology and identify that activation of oxidative stress and ER stress pathways is a critical avenue for PL anticancer effects.

Topics: Apoptosis; Cell Line, Tumor; Dioxolanes; Endoplasmic Reticulum Stress; Gene Expression Profiling; Humans; Oxidative Stress; Pancreatic Neoplasms

In this study, a novel concise series of molecules based on the structure of goniothalamin (1) was synthesized and evaluated against a highly metastatic human pancreatic cancer cell line (Panc-1). Among them, derivative 8 displayed a low IC50 value (2.7 μM) and its concentration for decreasing colony formation was 20-fold lower than goniothalamin (1). Both compounds reduced the levels of the receptor tyrosine kinase (AXL) and cyclin D1 which are known to be overexpressed in pancreatic cancer cells. Importantly, despite the fact that goniothalamin (1) and derivative 8 caused pancreatic cancer cell cycle arrest and cell death, only derivative 8 was able to downregulate pro-survival and proliferation pathways mediated by mitogen activated protein kinase ERK1/2. Another interesting finding was that Panc-1 cells treated with derivative 8 displayed a strong decrease in the transcription factor (c-Myc), hypoxia-inducible factor-1α (HIF-1α) and vascular endothelial growth factor (VEGF) protein levels. Notably, the molecular effects caused by derivative 8 might not be related to ROS generation, since no significant production of ROS was observed in low concentrations of this compound (from 1.5 up to 3 μM). Therefore, the downregulation of important mediators of pancreatic cancer aggressiveness by derivative 8 reveals its great potential for the development of new chemotherapeutic agents for pancreatic cancer treatment.

Topics: Acylation; Animals; Aza Compounds; Cell Line; Down-Regulation; Pancreatic Neoplasms; Pyrones; Signal Transduction