thapsigargin and Carcinoma--Non-Small-Cell-Lung

Mounting evidence indicates that activation of unfolded protein response (UPR) and metabolic reprogramming contribute to cancer cell migration and invasion, but the molecular mechanism of pro-EMT program through a coordinated action of UPR with metabolism has not been defined. In this study, we utilized ER stress-inducing reagent, thapsigargin (TG), to induced pharmacologic ER stress in lung cancer cells. Here. We report that the branch of UPR, IRE1α-XBP1 pathway plays a pivotal role in reprogramming lung cancer cell metabolism. At the molecular level, the expression of pyruvate dehydrogenase kinase-1 (PDK-1) is directly induced by XBP1 as a consequence of UPR activation, thus facilitating aerobic glycolysis and lactate production. We also demonstrated that PDK1 serves as a downstream element of UPR activation in induction of Snail and EMT program. In addition, PDK1-induced Snail was dependent on the lactate production derived from metabolic reprogramming. Our findings reveal a critical role of lactate in pro-invasion events and establishes a direct connection between ER-stress and metabolic reprogramming in facilitating cancer cell progression.

Topics: Carcinoma, Non-Small-Cell Lung; Endoplasmic Reticulum Stress; Endoribonucleases; Epithelial-Mesenchymal Transition; Humans; Lactates; Lung Neoplasms; Protein Serine-Threonine Kinases; Pyruvate Dehydrogenase Acetyl-Transferring Kinase; Thapsigargin; Unfolded Protein Response; X-Box Binding Protein 1

Pterostilbene (PT), the natural dimethylated analog of resveratrol (RSV), is a potent anticarcinogen for non-small-cell lung cancer (NSCLC), but its anti-NSCLC mechanisms remain unclear. In this study, we show that PT treatment time- and dose-dependently enhanced the endoplasmic reticulum stress (ERS) signaling (i.e., p-PERK, IRE1, ATF4, CHOP), thus decreasing the cell viability and inducing apoptosis in human PC9 and A549 NSCLC cell lines. Moreover, the decreased migratory and adhesive abilities, downregulation of intracellular glutathione (GSH) level, enhanced reactive oxygen species (ROS) generation, Caspase 3 activity and mitochondrial membrane depolarization were observed in NSCLC cells treated with PT. These effects were reversed by CHOP siRNA which inhibited the ERS signaling pathway, but were promoted by thapsigargin (a classical ERS inducer) in vitro. Besides, in vivo studies also verify that PT exerted anticancer activity by mobilizing ERS signaling and apoptosis-related proteins, and these effects were enhanced by thapsigargin. Therefore, ERS activation may represent a new mechanism of anti-NSCLC action by PT, and a novel therapeutic intervention for lung cancer.

Topics: A549 Cells; Animals; Antineoplastic Agents; Apoptosis; Apoptosis Regulatory Proteins; Carcinoma, Non-Small-Cell Lung; Cell Line; Cell Line, Tumor; Cell Survival; Down-Regulation; Endoplasmic Reticulum Stress; Humans; Lung Neoplasms; Male; Mice; Mice, Nude; Reactive Oxygen Species; RNA, Small Interfering; Signal Transduction; Stilbenes; Thapsigargin

Chemoresistance in cancer therapy is an unfavorable prognostic factor in non-small cell lung cancer (NSCLC). Elevation of intracellular calcium level in multidrug resistant (MDR) sublines leads to sensitization of MDR sublines to cell death. We demonstrated that a fungal protein from Ganoderma microsporum, GMI, elevates the intracellular calcium level and reduces the growth of MDR subline via autophagy and apoptosis, regardless of p-glycoprotein (P-gp) overexpression, in mice xenograft tumors. In addition, we examined the roles of autophagy in the death of MDR A549 lung cancer sublines by GMI, thapsigargin (TG) and tunicamycin (TM) in vitro. Cytotoxicity of TG was inhibited by overexpressed P-gp. However, TM-induced death of MDR sublines was independent of P-gp level. Combinations of TG and TM with either docetaxel or vincristine showed no additional cytotoxic effects on MDR sublines. TG- and TM-mediated apoptosis of MDR sublines was demonstrated on Annexin-V assay and Western blot and repressed by pan-caspase inhibitor (Z-VAD-FMK). Treatment of MDR sublines with TG and TM also augmented autophagy with accumulation of LC3-II proteins, breakdown of p62 and formation of acidic vesicular organelles (AVOs). Inhibition of ATG5 by shRNA silencing significantly reduced autophagy and cell death but not apoptosis following TG or TM treatment. GMI treatment inhibited the phosphorylation of Akt/S473 and p70S6K/T389. Interestingly, the phosphorylation of ERK was not associated with GMI-induced autophagy. We conclude that autophagy plays a pro-death role in acquired MDR and upregulation of autophagy by GMI via Akt/mTOR inhibition provides a potential strategy for overcoming MDR in the treatment of lung cancers.

Topics: Amino Acid Chloromethyl Ketones; Animals; Antineoplastic Agents; Apoptosis; ATP Binding Cassette Transporter, Subfamily B, Member 1; Autophagy; Autophagy-Related Protein 5; Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; Docetaxel; Drug Resistance, Multiple; Drug Resistance, Neoplasm; Fungal Proteins; Ganoderma; Humans; Lung Neoplasms; Male; Medicine, Chinese Traditional; Mice; Mice, Inbred NOD; Microtubule-Associated Proteins; Proto-Oncogene Proteins c-akt; Ribosomal Protein S6 Kinases, 70-kDa; RNA Interference; RNA, Small Interfering; Taxoids; Thapsigargin; TOR Serine-Threonine Kinases; Tunicamycin; Vincristine; Xenograft Model Antitumor Assays

The molecular carcinogenesis of lung cancer has yet to be clearly elucidated. We investigated the possible oncogenic function of SEC62 in lung cancer, which was predicted based on our previous findings that lung and thyroid cancer tissue samples exhibited increased Sec62 protein levels. The SEC62 gene locus is at 3q26.2, and 3q amplification is reportedly the most common genomic alteration in non-small cell lung cancer. We analyzed SEC62 mRNA and protein levels in tissue samples from lung cancer patients by real-time quantitative PCR, Western blot, and IHC and found significantly increased SEC62 mRNA and protein levels in tumors compared with tumor-free tissue samples from the same patients. Correlation analyses revealed significantly higher Sec62 levels in tumors with lymph node metastases compared with nonmetastatic tumors, as well as in poorly compared with moderately differentiated tumors. On the basis of these promising results, we examined the role of Sec62 in cancer cell biology in vitro. Cell migration assays with lung and thyroid cancer cells showed distinct stimulation of migration in SEC62-overexpressing cells and inhibition of migration in Sec62-depleted cells. Moreover, we found that SEC62 silencing sensitized the cells to thapsigargin-induced endoplasmic reticulum stress. Thus, our results indicate that SEC62 represents a potential candidate oncogene in the amplified 3q region in cases of non-small cell lung cancer and harbors various functions in cancer cell biology.

Topics: Adaptor Proteins, Signal Transducing; Blotting, Western; Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; Cell Movement; Chromosomes, Human, Pair 3; DNA Topoisomerases, Type II; DNA-Binding Proteins; Endoplasmic Reticulum Stress; Enzyme Inhibitors; Gene Amplification; Gene Silencing; Humans; Immunohistochemistry; Lung Neoplasms; Membrane Transport Proteins; Real-Time Polymerase Chain Reaction; RNA, Messenger; RNA, Small Interfering; Thapsigargin; Thyroid Neoplasms