Seung Un Seo

NOX4-mediated ROS production induces apoptotic cell death via down-regulation of c-FLIP and Mcl-1 expression in combined treatment with thioridazine and curcumin

Thioridazine is known to have anti-tumor effects by inhibiting PI3K/Akt signaling, which is an important signaling pathway in cell survival. However, thioridazine alone does not induce apoptosis in head and neck squamous cell carcinoma (AMC-HN4), human breast carcinoma (MDA-MB231), and human glioma (U87MG) cells. Therefore, we investigated whether combined treatment with thioridazine and curcumin induces apoptosis. Combined treatment with thioridazine and curcumin markedly induced apoptosis in cancer cells without inducing apoptosis in human normal mesangial cells and human normal umbilical vein cells (EA.hy926). We found that combined treatment with thioridazine and curcumin had synergistic effects in AMC-HN4 cells. Among apoptosis-related proteins, thioridazine plus curcumin induced down-regulation of c-FLIP and Mcl-1 expression at the post-translational levels in a proteasome-dependent manner. Augmentation of proteasome activity was related to the up-regulation of proteasome subunit alpha 5 (PSMA5) expression in curcumin plus thioridazine-treated cells. Combined treatment with curcumin and thioridazine produced intracellular ROS in a NOX4-dependent manner, and ROS-mediated activation of Nrf2/ARE signaling played a critical role in the up-regulation of PSMA5 expression. Furthermore, ectopic expression of c-FLIP and Mcl-1 inhibited apoptosis in thioridazine and curcumin-treated cells. Therefore, we demonstrated that thioridazine plus curcumin induces proteasome activity by up-regulating PSMA5 expression via NOX4-mediated ROS production and that down-regulation of c-FLIP and Mcl-1 expression post-translationally is involved in apoptosis.

Thioridazine enhances sensitivity to carboplatin in human head and neck cancer cells through downregulation of c-FLIP and Mcl-1 expression

Carboplatin is a less toxic analog of cisplatin, but carboplatin also has side effects, including bone marrow suppression. Therefore, to improve the capacity of the anticancer activity of carboplatin, we investigated whether combined treatment with carboplatin and thioridazine, which has antipsychotic and anticancer activities, has a synergistic effect on apoptosis. Combined treatment with carboplatin and thioridazine markedly induced caspase-mediated apoptosis in head and neck squamous cell carcinoma (AMC-HN4) cells. Combined treatment with carboplatin and thioridazine induced downregulation of Mcl-1 and c-FLIP expression. Ectopic expression of Mcl-1 and c-FLIP inhibited carboplatin plus thioridazine-induced apoptosis. We found that augmentation of proteasome activity had a critical role in downregulation of Mcl-1 and c-FLIP expression at the post-translational level in carboplatin plus thioridazine-treated cells. Furthermore, carboplatin plus thioridazine induced upregulation of the expression of proteasome subunit alpha 5 (PSMA5) through mitochondrial reactive oxygen species (ROS)-dependent nuclear factor E2-related factor 2 (Nrf2) activation. In addition, combined treatment with carboplatin and thioridazine markedly induced apoptosis in human breast carcinoma (MDA-MB231) and glioma (U87MG) cells, but not in human normal mesangial cells and normal human umbilical vein cells (EA.hy926). Collectively, our study demonstrates that combined treatment with carboplatin and thioridazine induces apoptosis through proteasomal degradation of Mcl-1 and c-FLIP by upregulation of Nrf2-dependent PSMA5 expression.

Z-FL-COCHO, a cathepsin S inhibitor, enhances oxaliplatin-mediated apoptosis through the induction of endoplasmic reticulum stress

Multiple cancer cells highly express cathepsin S, which has pro-tumoral effects. However, it was previously unknown whether knockdown or a pharmacological inhibitor (ZFL) of cathepsin S acts as an inducer of ER stress. Here, ZFL and knockdown of cathepsin S markedly induced ER stress through the up-regulation of calcium levels in the cytosol. Induction of calcium levels by inhibition of cathepsin S is markedly blocked by an inhibitor of the IP3 receptor and the ryanodine receptor Ca2+ channel in the ER, but an inhibitor of a mitochondrial Ca2+ uniporter had no effect on ZFL-induced calcium levels. Furthermore, production of mitochondrial ROS by ZFL was associated with an increase in cytosolic calcium levels. ZFL-mediated ER stress enhanced anti-cancer drug-induced apoptotic cell death, and pretreatment with chemical chaperones or down-regulation of ATF4 and CHOP by small interfering RNA markedly reduced ZFL plus oxaliplatin-induced apoptosis. Taken together, our findings reveal that inhibition of cathepsin S is an inducer of ER stress; these findings may contribute to the enhancement of therapeutic efficiency in cancer cells.Cancer: Enhancing sensitivity to anti-cancer drugsA drug that inhibits a key cancer enzyme could be used in combination with anti-cancer drugs to improve sensitivity to treatment. The intracellular endoplasmic reticulum (ER) is involved in several vital processes in cells, including folding and processing proteins. Taeg Kyu Kwon at Keimyung University, Daegu, South Korea, and co-workers have demonstrated how inhibition of cathepsin S, which is expressed in many cancer cells, induces ER stress. In trials on human kidney cancer cells grafted onto mice and in vitro, the team found that ZFL (cathepsin S inhibitor) triggered transient ER stress by increasing calcium levels inside cells. Subsequent treatment with the anti-cancer drug oxaliplatin resulted in increased cancer cell death.

mTORC1/2 inhibitor and curcumin induce apoptosis through lysosomal membrane permeabilization-mediated autophagy

AbstractmTOR is an important regulator of cell growth and forms two complexes, mTORC1/2. In cancer, mTOR signaling is highly activated, and the regulation of this signaling, as an anti-cancer strategy, has been emphasized. However, PP242 (inhibitor of mTORC1 and mTORC2) alone did not induce human renal carcinoma cell death. In this study, we found that PP242 alone did not alter cell viability, but combined curcumin and PP242 treatment induced cell death. Combined PP242 and curcumin treatment induced Bax activation and decreased expression of Mcl-1 and Bcl-2. Furthermore, co-treatment with PP242 and curcumin-induced the downregulation of the Rictor (an mTORC2 complex protein) and Akt protein levels, and ectopic overexpression of Rictor or Akt inhibited PP242 plus curcumin induced cell death. Downregulation of Rictor increased cytosolic Ca2+ release from endoplasmic reticulum, which led to lysosomal damage in PP242 plus curcumin-treated cells. Furthermore, damaged lysosomes induced autophagy. Autophagy inhibitors markedly inhibited cell death. Finally, combined curcumin and PP242 treatment reduced tumor growth and induced cell death in xenograft models. Altogether, our results reveal that combined PP242 and curcumin treatment could induce autophagy-mediated cell death by reducing the expression of Rictor and Akt in renal carcinoma cells.

Angelicin potentiates TRAIL-induced apoptosis in renal carcinoma Caki cells through activation of caspase 3 and down-regulation of c-FLIP expression

Preclinical Research & Development

Up-regulation of 5-lipoxygenase by inhibition of cathepsin G enhances TRAIL-induced apoptosis through down-regulation of survivin

Cathepsin G is a serine protease secreted from activated neutrophils, it has important roles in inflammation and immune response. Moreover, cathepsin G promotes tumor cell-cell adhesion and migration in cancer cells. In this study, we investigated whether inhibition of cathepsin G could sensitize TRAIL-mediated apoptosis in cancer cells. An inhibitor of cathepsin G [Cathepsin G inhibitor I (Cat GI); CAS 429676-93-7] markedly induced TRAIL-mediated apoptosis in human renal carcinoma (Caki, ACHN, and A498), lung cancer (A549) and cervical cancer (Hela) cells. In contrast, combined treatment with Cat GI and TRAIL had no effect on apoptosis in normal cells [mesangial cell (MC) and human skin fibroblast (HSF)]. Cat GI induced down-regulation of survivin expression at the post-translational level, and overexpression of survivin markedly blocked apoptosis induced by combined treatment with Cat GI plus TRAIL. Interestingly, Cat GI induced down-regulation of survivin via 5-lipoxygenase (5-LOX)-mediated reactive oxygen species (ROS) production. Inhibition of 5-LOX by gene silencing (siRNA) or a pharmacological inhibitor of 5-LOX (zileuton) markedly attenuated combined treatment-induced apoptosis. Taken together, our results indicate that inhibition of cathepsin G sensitizes TRAIL-induced apoptosis through 5-LOX-mediated down-regulation of survivin expression.

Volasertib Enhances Sensitivity to TRAIL in Renal Carcinoma Caki Cells through Downregulation of c-FLIP Expression

Polo-like kinase 1 (PLK1) plays major roles in cell cycle control and DNA damage response. Therefore, PLK1 has been investigated as a target for cancer therapy. Volasertib is the second-in class dihydropteridinone derivate that is a specific PLK1 inhibitor. In this study, we examined that combining PLK1 inhibitor with tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) would have an additive and synergistic effect on induction of apoptosis in cancer cells. We found that volasertib alone and TRAIL alone had no effect on apoptosis, but the combined treatment of volasertib and TRAIL markedly induced apoptosis in Caki (renal carcinoma), A498 (renal carcinoma) and A549 (lung carcinoma) cells, but not in normal cells (human skin fibroblast cells and mesangial cells). Combined treatment induced accumulation of sub-G1 phase, DNA fragmentation, cleavage of poly (ADP-ribose) polymerase (PARP) and activation of caspase 3 activity in Caki cells. Interestingly, combined treatment induced downregulation of cellular-FLICE-inhibitory protein (c-FLIP) expression and ectopic expression of c-FLIP markedly blocked combined treatment-induced apoptosis. Therefore, this study demonstrates that volasertib may sensitize TRAIL-induced apoptosis in Caki cells via downregulation of c-FLIP.

Kv3.1 and Kv3.4, Are Involved in Cancer Cell Migration and Invasion

Voltage-gated potassium (Kv) channels, including Kv3.1 and Kv3.4, are known as oxygen sensors, and their function in hypoxia has been well investigated. However, the relationship between Kv channels and tumor hypoxia has yet to be investigated. This study demonstrates that Kv3.1 and Kv3.4 are tumor hypoxia-related Kv channels involved in cancer cell migration and invasion. Kv3.1 and Kv3.4 protein expression in A549 and MDA-MB-231 cells increased in a cell density-dependent manner, and the pattern was similar to the expression patterns of hypoxia-inducible factor-1α (HIF-1α) and reactive oxygen species (ROS) according to cell density, whereas Kv3.3 protein expression did not change in A549 cells with an increase in cell density. The Kv3.1 and Kv3.4 blocker blood depressing substance (BDS) did not affect cell proliferation; instead, BDS inhibited cell migration and invasion. We found that BDS inhibited intracellular pH regulation and extracellular signal-regulated kinase (ERK) activation in A549 cells cultured at a high density, potentially resulting in BDS-induced inhibition of cell migration and invasion. Our data suggest that Kv3.1 and Kv3.4 might be new therapeutic targets for cancer metastasis.

Cepharanthine Enhances TRAIL-Mediated Apoptosis Through STAMBPL1-Mediated Downregulation of Survivin Expression in Renal Carcinoma Cells

Cepharanthine (CEP) is a natural plant alkaloid, and has anti-inflammatory, antineoplastic, antioxidative and anticancer properties. In this study, we investigated whether CEP could sensitize renal carcinoma Caki cells to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis. CEP alone and TRAIL alone had no effect on apoptosis. However, combined CEP and TRAIL treatment markedly enhanced apoptotic cell death in cancer cells, but not in normal cells. CEP induced downregulation of survivin and cellular-FLICE inhibitory protein (c-FLIP) expression at post-translational levels. Ectopic expression of survivin blocked apoptosis by combined treatment with CEP plus TRAIL, but not in c-FLIP overexpression. Interestingly, CEP induced survivin downregulation through downregulation of deubiquitin protein of STAM-binding protein-like 1 (STAMBPL1). Overexpression of STAMBPL1 markedly recovered CEP-mediated survivin downregulation. Taken together, our study suggests that CEP sensitizes TRAIL-mediated apoptosis through downregulation of survivin expression at the post-translational levels in renal carcinoma cells.

BIX-01294 sensitizes renal cancer Caki cells to TRAIL-induced apoptosis through downregulation of survivin expression and upregulation of DR5 expression

BIX-01294 (BIX), a G9a histone methyltransferase inhibitor, has been reported for its anti-proliferative and anticancer activities against various cancer cell lines. In this study, we investigated whether BIX could sensitize TRAIL-mediated apoptosis in various cancer cells. Combined treatment with BIX and TRAIL markedly induced apoptosis in human renal carcinoma (Caki, ACHN, and A498), breast carcinoma (MCF-7), and lung carcinoma (A549) cells. In contrast, BIX and TRAIL co-treatment did not induce apoptosis in normal cells, specifically mouse kidney cell (TCMK-1) and human skin fibroblast (HSF). BIX downregulated protein expression levels of XIAP and survivin at the post-translational level. Overexpression of survivin markedly blocked combined BIX and TRAIL treatment-induced apoptosis, but XIAP had no effect. Furthermore, BIX induced upregulation of DR5 expression at the transcriptional levels, and knockdown of DR5 expression using small interfering RNAs (siRNAs) markedly attenuated BIX and TRAIL-induced apoptosis. Interestingly, siRNA-mediated G9a histone methyltransferase knockdown also enhanced TRAIL-induced apoptosis in Caki cells. However, knockdown of G9a did not change expression levels of XIAP, survivin, and DR5. Therefore, BIX-mediated TRAIL sensitization was independent of histone methyltransferase G9a activity. Taken together, these results suggest that BIX facilitates TRAIL-mediated apoptosis via downregulation of survivin and upregulation of DR5 expression in renal carcinoma Caki cells.▶ BIX facilitates TRAIL-mediated apoptosis in human renal carcinoma Caki cells.▶ Downregulation of survivin contributes to BIX plus TRAIL-induced apoptosis.▶ Upregulation of DR5 is involved in BIX plus TRAIL-mediated apoptosis.▶ BIX-mediated TRAIL sensitization is independent of ROS production.