ABSTRACT
Exploiting protein homeostasis is a new therapeutic approach in cancer. Nelfinavir (NFV) is an HIV protease inhibitor that induces endoplasmic reticulum (ER) stress in cancer cells. Under conditions of ER stress, misfolded proteins are transported from the ER back to the cytosol for subsequent degradation by the ubiquitin-proteasome system. Bortezomib (BZ) is a proteasome inhibitor and interferes with degradation of misfolded proteins. Here, we show that NFV and BZ enhance proteotoxicity in non-small cell lung cancer (NSCLC) and multiple myeloma (MM) cells. The combination synergistically inhibited cell proliferation and induced cell death. Activating transcription factor (ATF)3 and CCAAT-enhancer binding protein homologous protein (CHOP), markers of ER stress, were rapidly increased, and their siRNA-mediated knockdown inhibited cell death. Knockdown of double-stranded RNA activated protein kinase-like ER kinase, a signal transducer in ER stress, significantly decreased apoptosis. Pretreatment with the protein synthesis inhibitor, cycloheximide, decreased levels of ubiquitinated proteins, ATF3, CHOP, and the overall total cell death, suggesting that inhibition of protein synthesis increases cell survival by relieving proteotoxic stress. The NFV/BZ combination inhibited the growth of NSCLC xenografts, which correlated with the induction of markers of ER stress and apoptosis. Collectively, these data show that NFV and BZ enhance proteotoxicity in NSCLC and MM cells, and suggest that this combination could tip the precarious balance of protein homeostasis in cancer cells for therapeutic gain.
Subject(s)
Boronic Acids/toxicity , Nelfinavir/toxicity , Protease Inhibitors/toxicity , Pyrazines/toxicity , Activating Transcription Factor 3/antagonists & inhibitors , Activating Transcription Factor 3/genetics , Activating Transcription Factor 3/metabolism , Animals , Apoptosis/drug effects , Boronic Acids/therapeutic use , Bortezomib , CCAAT-Enhancer-Binding Proteins/antagonists & inhibitors , CCAAT-Enhancer-Binding Proteins/genetics , CCAAT-Enhancer-Binding Proteins/metabolism , Carcinoma, Non-Small-Cell Lung/drug therapy , Carcinoma, Non-Small-Cell Lung/metabolism , Carcinoma, Non-Small-Cell Lung/pathology , Caspases/metabolism , Cell Line, Tumor , Cell Proliferation/drug effects , Drug Synergism , Endoplasmic Reticulum Stress/drug effects , Humans , Mice , Mice, Nude , Multiple Myeloma/drug therapy , Multiple Myeloma/metabolism , Multiple Myeloma/pathology , Nelfinavir/therapeutic use , Protease Inhibitors/therapeutic use , Pyrazines/therapeutic use , RNA Interference , RNA, Small Interfering/metabolism , Transplantation, HeterologousABSTRACT
Anticancer phospholipids that inhibit Akt such as the alkylphospholipid perifosine (Per) and phosphatidylinositol ether lipid analogs (PIAs) promote cellular detachment and apoptosis and have a similar cytotoxicity profile against cancer cell lines in the NCI60 panel. While investigating the mechanism of Akt inhibition, we found that short-term incubation with these compounds induced rapid shedding of cellular nanovesicles containing EGFR, IGFR and p-Akt that occurred in vitro and in vivo, while prolonged incubation led to cell detachment and death that depended on sphingomyelinase-mediated generation of ceramide. Pretreatment with sphingomyelinase inhibitors blocked ceramide generation, decreases in phospho-Akt, nanovesicle release and cell detachment in response to alkylphospholipids and PIAs in non-small cell lung cancer cell lines. Similarly, exogenous ceramide also decreased active Akt and induced nanovesicle release. Knockdown of neutral sphingomyelinase decreased, whereas overexpression of neutral or acid sphingomyelinase increased cell detachment and death in response to the compounds. When transferred in vitro, PIA or Per-induced nanovesicles increased ceramide levels and death in recipient cells. These results indicate ceramide generation underlies the Akt inhibition and cytotoxicity of this group of agents, and suggests nanovesicle shedding and uptake might potentially propagate their cytotoxicity in vivo.
Subject(s)
Antineoplastic Agents/toxicity , Apoptosis/drug effects , Ceramides/metabolism , Phosphatidylinositol Phosphates/toxicity , Phosphorylcholine/analogs & derivatives , Secretory Vesicles/metabolism , Animals , Cell Line, Tumor , ErbB Receptors/metabolism , Female , Humans , Lung Neoplasms/drug therapy , Lung Neoplasms/metabolism , Lung Neoplasms/pathology , Mice , Mice, Nude , Phosphorylcholine/chemistry , Phosphorylcholine/therapeutic use , Phosphorylcholine/toxicity , Proto-Oncogene Proteins c-akt/antagonists & inhibitors , Proto-Oncogene Proteins c-akt/metabolism , Pyridines/therapeutic use , Pyridines/toxicity , RNA Interference , RNA, Small Interfering/metabolism , Receptor, IGF Type 1/metabolism , Sphingomyelin Phosphodiesterase/antagonists & inhibitors , Sphingomyelin Phosphodiesterase/genetics , Sphingomyelin Phosphodiesterase/metabolism , Transplantation, HeterologousABSTRACT
Nonmelanoma skin cancer afflicts more than one million people in the U.S. annually, highlighting the need for more effective preventive regimens. We have investigated the ability of deguelin, a plant-derived rotenoid with cancer chemopreventive activity, to inhibit UVB-induced skin carcinogenesis with the SKh-1 mouse model. Topically-applied deguelin significantly inhibited the multiplicity of UVB-induced skin tumors, indicating potential as a human skin cancer chemopreventive agent. Mechanistic studies to determine the potential of deguelin to block a number of established UVB-induced molecular events yielded negative results [including UVB-induced AP-1 DNA binding, c-fos and TNFalpha mRNA induction, arachidonic acid release and UVB-induced phosphorylation of mTOR (Ser2448), akt (Ser473) and erk (Thr202/Tyr204)]. These results are of interest as they contradict a major hypothesis for the mode of action of deguelin, i.e., a general down regulation of signal transduction based on inhibition of NADH dehydrogenase and depletion of ATP levels. In the current work, however, deguelin was found to activate 5' AMP-activated kinase (AMPK), a protein that acts as a cellular energy sensor. This is the first report of a chemopreventive agent having this effect and suggests a possible role for AMPK in cancer chemoprevention.
Subject(s)
Multienzyme Complexes/metabolism , Protein Serine-Threonine Kinases/metabolism , Rotenone/analogs & derivatives , Skin Neoplasms/prevention & control , Ultraviolet Rays/adverse effects , AMP-Activated Protein Kinases , Adenosine Triphosphate/metabolism , Animals , Chemoprevention , DNA Adducts , Down-Regulation , Female , Humans , Mice , Rotenone/pharmacology , Signal Transduction , Skin Neoplasms/etiology , Tumor Cells, CulturedABSTRACT
Two new cardenolides, (-)-14-hydroxy-3beta-(3-O-methyl-6-deoxy-alpha-L-rhamnosyl)-11a lpha, 12alpha-epoxy-(5beta,14beta,17betaH)-card-20 (22)-enolide (1), (-)-14-hydroxy-3beta-(3-O-methyl-6-deoxy-alpha-L-glucopyranosyl)-11al pha,12alpha-epoxy-(5beta,14beta,17betaH)-card -20(22)-enolide (2), and a known cardenolide, (-)-17beta-neriifolin (3), were isolated from the roots of Cerbera manghas as antiproliferative and antiestrogenic principles when evaluated against a human colon cancer cell line (Col2) and the Ishikawa cell line, respectively. Two known lignans, (-)-olivil (4) and (-)-cycloolivil (5), were also isolated but were inactive in the assay systems used.
