CASP4

Gene Summary

Gene:CASP4; caspase 4
Aliases: TX, Mih1, ICH-2, Mih1/TX, ICEREL-II, ICE(rel)II
Location:11q22.3
Summary:This gene encodes a protein that is a member of the cysteine-aspartic acid protease (caspase) family. Sequential activation of caspases plays a central role in the execution-phase of cell apoptosis. Caspases exist as inactive proenzymes composed of a prodomain and a large and small protease subunit. Activation of caspases requires proteolytic processing at conserved internal aspartic residues to generate a heterodimeric enzyme consisting of the large and small subunits. This caspase is able to cleave and activate its own precursor protein, as well as caspase 1 precursor. When overexpressed, this gene induces cell apoptosis. Alternative splicing results in transcript variants encoding distinct isoforms. [provided by RefSeq, Jul 2008]
Databases:VEGA, OMIM, HGNC, Ensembl, GeneCard, Gene
Protein:caspase-4
Source:NCBIAccessed: 09 March, 2017

Ontology:

What does this gene/protein do?
Show (6)
Pathways:What pathways are this gene/protein implicaed in?
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Cancer Overview

Research Indicators

Publications Per Year (1992-2017)
Graph generated 10 March 2017 using data from PubMed using criteria.

Literature Analysis

Mouse over the terms for more detail; many indicate links which you can click for dedicated pages about the topic.

  • bcl-2-Associated X Protein
  • CASP4
  • Estrogen Receptors
  • Western Blotting
  • Apoptosis
  • Single-Stranded Conformational Polymorphism
  • Caspase 3
  • Breast Cancer
  • Proteins
  • eIF-2 Kinase
  • Tunicamycin
  • Caspases
  • Cell Proliferation
  • Unfolded Protein Response
  • Cell Survival
  • Antineoplastic Agents
  • Thiocarbamates
  • Messenger RNA
  • Oligonucleotide Array Sequence Analysis
  • Proto-Oncogene Proteins
  • Caspases, Initiator
  • Tyrosine
  • Endoplasmic Reticulum Stress
  • Ovarian Cancer
  • beta Catenin
  • Stilbenes
  • Endoplasmic Reticulum
  • Mitochondria
  • Enzyme Activation
  • Cancer Gene Expression Regulation
  • Chromosome 11
  • Gene Expression Profiling
  • Calcium
  • DNA-Binding Proteins
  • Colorectal Cancer
  • Heat-Shock Proteins
  • Sulfides
  • Up-Regulation
  • Peptides, Cyclic
  • Vitamin E
  • Cervical Cancer
  • Transcription Factor AP-1
  • Oxidative Stress
Tag cloud generated 09 March, 2017 using data from PubMed, MeSH and CancerIndex

Specific Cancers (4)

Data table showing topics related to specific cancers and associated disorders. Scope includes mutations and abnormal protein expression.

Note: list is not exhaustive. Number of papers are based on searches of PubMed (click on topic title for arbitrary criteria used).

Latest Publications: CASP4 (cancer-related)

Zheng LC, Yang MD, Kuo CL, et al.
Norcantharidin-induced Apoptosis of AGS Human Gastric Cancer Cells Through Reactive Oxygen Species Production, and Caspase- and Mitochondria-dependent Signaling Pathways.
Anticancer Res. 2016; 36(11):6031-6042 [PubMed] Related Publications
Norcantharidin (NCTD) was purified from mylabris, the dried body of the Chinese blister beetle. NCTD has been shown to exhibit anticancer activities in many human cancer cell lines, but there are no reports to show whether it induces apoptosis of human gastric cancer cells. Therefore, in the present study, we investigated NCTD-induced cell death and associated protein expression in human gastric cancer AGS cells in vitro. Cell morphological changes, viability and cell-cycle distribution were examined and analyzed by phase-contrast microscopy and flow cytometric assays. Flow cytometry was also used to measure the levels of reactive oxygen species (ROS), Ca(2+), mitochondrial membrane potential (Ψm) and activity of caspases. The results indicated that NCTD induced cell morphological changes, reduced total viable cell number and induced G0/G1 phase arrest. NCTD also increased ROS production and reduced the Ψm and increased caspase-9 activity in AGS cells. Western blotting also found that NCTD increased the pro-apoptotic proteins such as BCL2-associated X protein (BAX) and BH3 interacting-domain death agonist (BID) and increased the release of cytochrome c, apoptosis inducing factor (AIF) and endonuclease G (Endo G) release from mitochondria in AGS cells. NCTD also significantly increased the expression of active forms of caspase-3 and -8 and -9 and reduced the expression of caspase-4 and -12 in AGS cells. Based on these observations, we suggest that NCTD-induced apoptotic cell death may be through mitochondria- and caspase-dependent pathways.

Zhu YQ, Wang BY, Wu F, et al.
Influence of Tanshinone IIA on the Apoptosis of Human Esophageal Ec-109 Cells.
Nat Prod Commun. 2016; 11(1):17-9 [PubMed] Related Publications
The induced-apoptosis effect and mechanism of human esophageal cancer Ec-109 cells via tanshinone IIA was investigated. The Ec-109 cells were cultured in vitro with different concentrations of tanshinone IIA (2 µg/mL, 4 µg/mL, or 8 µg/mL) for 12, 24, 36, and 48 hours. MTT assay was used to evaluate the proliferative inhibition rate of tanshinone IIA on esophageal Ec-109 cells. After 24 hours of culturing in vitro, a control group was assigned. The apoptosis rate was detected by the AO/EB and annexin V-FITC/propidium iodide assay, and the protein levels of Caspase-4 and CHOP were determined by the Western blot technique. MTT data showed that tanshinone IIA could significantly inhibit the proliferation of Ec-109 cells with a dose- and time-dependent mode. Compared with the control group, tanshinone IIA could apparently induce apoptosis of Ec-109 cells, and the level of Caspase-4 and CHOP (p < 0.01) obviously increased. Tanshinone IIA can significantly induce the apoptosis of Ec-109 cells, which may take effect by the stress pathway of the endoplasmic reticulum.

Yang F, Tang XY, Liu H, Jiang ZW
Inhibition of mitogen-activated protein kinase signaling pathway sensitizes breast cancer cells to endoplasmic reticulum stress-induced apoptosis.
Oncol Rep. 2016; 35(4):2113-20 [PubMed] Related Publications
Accumulation of unfolded proteins in the endoplasmic reticulum (ER) induces ER stress which is observed in many human diseases, including breast cancer. Cellular adaptation to ER stress is mediated by the unfolded protein response (UPR), which aims at restoring ER homeostasis. Higher levels of GRP78 expression indicates constitutive activation of the UPR in breast cancer leading to breast cancer cells that are relatively resistant to ER stress-induced apoptosis. Tunicamycin (TM), an ER stress inducer, constitutively activates the mitogen-activated protein kinase (MAPK)/extracellular signal regulated kinase (ERK), and (MEK)/ERK pathway which plays a role in upregulation of GRP78 by ER stress in that inhibition of MEK by U0126 reduces the levels of GRP78 and blocks its upregulation by TM. Inhibition of the MEK/ERK pathway by U0126 sensitizes breast cancer cells to TM-induced apoptosis. Inhibition of GRP78 by siRNA knockdown enhances TM- and U0126-induced apoptosis in breast cancer cells. This sensitization of breast cancer cells to TM-induced apoptosis by inhibition of MEK/ERK and GRP78 is caspase-dependent, at least in part, by activation of caspase-4. These results seem to indicate that GRP78 has potential as a chemotherapeutical target and have important implications for new treatment strategies in breast cancer by combination with agents that induce ER stress with inhibitors of the MEK/ERK pathway.

Li S, Wang SM, Yang YB, Liu QO
[Effect of Viqi Chutan Recipe on Caspase-4 and DNA-PK of Cell Apoptosis Approach in Transplanted Lung Cancer A549 Cells in Nude Mice].
Zhong Yao Cai. 2015; 38(6):1247-50 [PubMed] Related Publications
OBJECTIVE: To study the inhibitory effect of Yiqi Chutan Recipe on the transplanted tumor through endoplasmic reticulum UPR-mediated approach.
METHODS: 40 lung cancer A549 cells models transplanted in nude mice were established. On the 7th day of inoculation, mice were randomly divided into model group( saline group) , Cisplatin group (0.002 g/kg), Yiqi Chutan Recipe low dose group (3.0 g/kg), Yiqi Chutan Recipe high dose group(6. 0 g/kg)and Yiqi Chutan Recipe (3.0 g/kg)with Cisplatin group (0.002 g/kg). Each aforementioned group had eight mice. Mice were treated by Yiqi Chutan Recipe to gavage one time a day, for 21 days, and by Cisplatin Injection to intraperitoneal injection one time a day, for 7 days. On the 22th day, all mice were executed to death. Then each tumor's weight and volume were measured, and the expression of Caspase-4 and DNA-PK protein were detected through immunohistochemical method and Western blot method.
RESULTS: Compared with model group, the tumors' volume and weight of Yiqi Chutan Recipe high dose group and Yiqi Chutan Recipe with Cisplatin group were decreased, but the expressions of Caspase-4 and DNA-PK protein in tumors were increased (P < 0.01). Yiqi Chutan Recipe with Cisplatin Group had the better effect (P < 0.05).
CONCLUSION: Yiqi Chutan Recipe has a certain inhibitory effect on A549 lung cancer in mice and its possible mechanism is relevant to the increase of expression of Caspase-4 and DNA-PK protein.

Qu X, Fan L, Zhong T, et al.
The nematocysts venom of Chrysaora helvola Brandt leads to apoptosis-like cell death accompanied by uncoupling of oxidative phosphorylation.
Toxicon. 2016; 110:74-8 [PubMed] Related Publications
The present work investigated the effects of the nematocysts venom (NV) from the Chrysaora helvola Brandt (C. helvola) jellyfish on the human nasopharyngeal carcinoma cell line, CNE-2. The medium lethal concentration (LC50), quantified by MTT assays, was 1.7 ± 0.53 μg/mL (n = 5). An atypical apoptosis-like cell death was confirmed by LDH release assay and Annexin V-FITC/PI staining-based flow cytometry. Interestingly, activation of caspase-4 other than caspase-3, -8, -9 and -1 was observed. Moreover, the NV stimuli caused a time-dependent loss of mitochondrial membrane potential (ΔΨm) as was an intracellular ROS burst. These results indicated that there was uncoupling of oxidative phosphorylation (UOP). An examination of the intracellular pH value by a pH-sensitive fluorescent probe, BCECF, suggested that the UOP was due to the time-dependent increase in the intracellular pH. This is the first report that jellyfish venom can induce UOP.

El-Khattouti A, Selimovic D, Hannig M, et al.
Imiquimod-induced apoptosis of melanoma cells is mediated by ER stress-dependent Noxa induction and enhanced by NF-κB inhibition.
J Cell Mol Med. 2016; 20(2):266-86 [PubMed] Free Access to Full Article Related Publications
Melanoma is characterized by dysregulated intracellular signalling pathways including an impairment of the cell death machinery, ultimately resulting in melanoma resistance, survival and progression. This explains the tumour's extraordinary resistance to the standard treatment. Imiquimod is a topical immune response modifier (imidazoquinoline) with both antiviral and antitumour activities. The mechanism by which imiquimod triggers the apoptosis of melanoma cells has now been carefully elucidated. Imiquimod-induced apoptosis is associated with the activation of apoptosis signalling regulating kinase1/c-Jun-N-terminal kinase/p38 pathways and the induction of endoplasmic stress characterized by the activation of the protein kinase RNA-like endoplasmic reticulum kinase signalling pathway, increase in intracellular Ca(2+) release, degradation of calpain and subsequent cleavage of caspase-4. Moreover, imiquimod triggers the activation of NF-κB and the expression of the inhibitor of apoptosis proteins (IAPs) such as, X-linked IAP (XIAP) together with the accumulation of reactive oxygen species (ROS). Also, imiquimod triggers mitochondrial dysregulation characterized by the loss of mitochondrial membrane potential (Δψm), the increase in cytochrome c release, and cleavage of caspase-9, caspase-3 and poly(ADP-ribose) polymerase (PARP). Inhibitors of specific pathways, permit the elucidation of possible mechanisms of imiquimod-induced apoptosis. They demonstrate that inhibition of NF-kB by the inhibitor of nuclear factor kappa-B kinase (IKK) inhibitor Bay 11-782 or knockdown of XIAP induces melanoma apoptosis in cells exposed to imiquimod. These findings support the use of either IKK inhibitors or IAP antagonists as adjuvant therapies to improve the effectiveness topical imiquimod in the treatment of melanoma.

Klieser E, Illig R, Státtner S, et al.
Endoplasmic Reticulum Stress in Pancreatic Neuroendocrine Tumors is Linked to Clinicopathological Parameters and Possible Epigenetic Regulations.
Anticancer Res. 2015; 35(11):6127-36 [PubMed] Related Publications
BACKGROUND: Endoplasmic reticulum (ER) stress is a highly-conserved cellular defense mechanism in response to perturbations of ER function. The role of ER stress in pancreatic neuroendocrine tumors (pNET) still remains unclear.
MATERIALS AND METHODS: We analyzed the protein expression pattern of the four key players of ER stress, (chaperone binding imunoglobluin protein (BiP), C/EBP homologous protein (CHOP), activating transcription factor 4 (ATF4) and caspase 4) as well as histone deacetylases (HDACs) by a tissue microarray (TMA) of 49 human pNET resected between 1997 and 2013 following, extensive clinicopathological characterization.
RESULTS: Immunohistochemical profiling revealed a significant up-regulation of BiP, ATF4, CHOP and caspase 4 in pNET cases compared to normal controls. Correlated to clinicopathological parameters especially BiP expression could be linked to higher grading and proliferation as well as to lower survival probability. Finally, expression of ER stress markers correlated with HDAC expression in situ and pharmalogical inhibition by panobinostat significantly reduced cell viability in vitro.
CONCLUSION: Up-regulation of ER stress in pNET indicates the presence and engagement of ER stress signaling in this tumor entity demonstrating another possible anticancer therapy option in pNET.

Wang LL, Hu RC, Dai AG, Tan SX
Bevacizumab induces A549 cell apoptosis through the mechanism of endoplasmic reticulum stress in vitro.
Int J Clin Exp Pathol. 2015; 8(5):5291-9 [PubMed] Free Access to Full Article Related Publications
AIMS: To observe the effect of bevacizumab on human A549 cells and explore its mechanism.
METHODS: After different concentrations (0 μM, 1 μM, 5 μM, 25 μM) of bevacizumab treating in A549 cells, CCK8 assay detect the impact of bevacizumab on A549 cell proliferation and flow cytometry determine the effect of bevacizumab on human A549 cells apoptosis. Real-time PCR and Western blotting detect the changing expression of the target gene (CHOP, caspase-4, IRE1, XBP-1) on mRNA and Protein level.
RESULTS: Treatment with bevacizumab for 24-hr have induced cell death in a does-dependent manner dramatically (P<0.05). In terms of the mRNA level, expression of XBP-1 has increased obviously in each group (1 μM, 5 μM, 25 μM) (P<0.01); the expression of CHOP (25 μM) and caspase-4 (5 μM) have increased slightly (P<0.05). In terms of the protein level, the expression of CHOP has increased obviously in each group (1 μM, 5 μM, 25 μM) when compared with the control group (0 μM) (P<0.05). As for caspase-4 (5 μM, 25 μM), the expression have increased slightly when compared with the control group (0 μM) (P<0.05).
CONCLUSION: Bevacizumab can induce A549 cell apoptosis through the mechanism of endoplasmic reticulum stress.

Flood B, Oficjalska K, Laukens D, et al.
Altered expression of caspases-4 and -5 during inflammatory bowel disease and colorectal cancer: Diagnostic and therapeutic potential.
Clin Exp Immunol. 2015; 181(1):39-50 [PubMed] Free Access to Full Article Related Publications
Caspases are a group of proteolytic enzymes involved in the co-ordination of cellular processes, including cellular homeostasis, inflammation and apoptosis. Altered activity of caspases, particularly caspase-1, has been implicated in the development of intestinal diseases, such as inflammatory bowel disease (IBD) and colorectal cancer (CRC). However, the involvement of two related inflammatory caspase members, caspases-4 and -5, during intestinal homeostasis and disease has not yet been established. This study demonstrates that caspases-4 and -5 are involved in IBD-associated intestinal inflammation. Furthermore, we found a clear correlation between stromal caspase-4 and -5 expression levels, inflammation and disease activity in ulcerative colitis patients. Deregulated intestinal inflammation in IBD patients is associated with an increased risk of developing CRC. We found robust expression of caspases-4 and -5 within intestinal epithelial cells, exclusively within neoplastic tissue, of colorectal tumours. An examination of adjacent normal, inflamed and tumour tissue from patients with colitis-associated CRC confirmed that stromal expression of caspases-4 and -5 is increased in inflamed and dysplastic tissue, while epithelial expression is restricted to neoplastic tissue. In addition to identifying caspases-4 and -5 as potential targets for limiting intestinal inflammation, this study has identified epithelial-expressed caspases-4 and -5 as biomarkers with diagnostic and therapeutic potential in CRC.

Zhang R, Wang R, Chen Q, Chang H
Inhibition of autophagy using 3-methyladenine increases cisplatin-induced apoptosis by increasing endoplasmic reticulum stress in U251 human glioma cells.
Mol Med Rep. 2015; 12(2):1727-32 [PubMed] Free Access to Full Article Related Publications
Cisplatin is one of the most widely used chemotherapeutic drugs; however, the side effects and drug resistance limit its usage. Previous findings have demonstrated that cisplatin kills tumor cells through endoplasmic reticulum (ER) stress, which provides a novel method to minimize cisplatin toxicity and circumvent cisplatin resistance. ER stress induces cell autophagy, cell apoptosis and the complicated regulatory network between them. The role of autophagy in cisplatin chemotherapy remains to be elucidated. 3-Methyladenine (3-MA) is normally used as an inhibitor of autophagy. The present study reveals a significant role of the inhibition of autophagy by treatment with 3-MA and cisplatin in combination in U251 human glioma cells. It was demonstrated that cisplatin induced the ER stress associated with apoptosis and autophagy in U251 cells. Inhibition of autophagy by 3-MA increased the expression levels of protein disulfide isomerase, ubiquitinated proteins, glucose regulated protein 78 and CCAAT-enhancer-binding protein homologous protein, and induced the activation of caspase-4 and caspase-3. Treatment with 3-MA combined with cisplatin increased cisplatin-induced apoptosis by increasing ER stress. Therefore, the inhibition of autophagy has the potential to improve cisplatin chemotherapy.

Ko YC, Lien JC, Liu HC, et al.
Demethoxycurcumin induces the apoptosis of human lung cancer NCI-H460 cells through the mitochondrial-dependent pathway.
Oncol Rep. 2015; 33(5):2429-37 [PubMed] Related Publications
Lung cancer is the most common cause of cancer-related mortality in the US as well as other regions of the world. Curcumin, demethoxycurcumin (DMC) and bisdemethoxycurcumin (BDMC) are the major components of Curcuma longa L. It has been reported that curcumin inhibits the growth of various types of cancer cells in vitro and in vivo. However, the mechanisms involved in the inhibition of cell growth and induced apoptosis by DMC in human lung cancer cells remain unclear. In the present study, we investigated the effect of DMC on cell death via the induction of apoptosis in NCI-H460 human lung cancer cells. Flow cytometric assay was used to examine the total percentage of viable cells, the population of cells in the sub-G1 phase of the cell cycle, the level of reactive oxygen species (ROS), Ca²⁺ production, mitochondrial membrane potential (ΔΨm) and caspase activity. Western blotting was used to examine the changes in the expression of cell cycle- and apoptosis-associated proteins. Confocal microscopy was used to examine the translocation of apoptosis-associated proteins. The results indicated that DMC significantly induced cell morphological changes and decreased the percentage of viable NCI-H460 cells and DMC induced apoptosis based on the cell distribution in the sub-G1 phase. Moreover, DMC promoted ROS and Ca²⁺ production and decreased the level of ΔΨm and promoted the activities of caspase-3, -8 and -9. The Western blotting results showed that DMC promoted the expression of AIF, Endo G and PARP. The levels of Fas ligand (Fas L) and Fas were also upregulated. Furthermore, DMC promoted expression of ER stress-associated proteins such as GRP78, GADD153, IRE1β, ATF-6α, ATF-6β and caspase-4. Based on the findings, we suggest that DMC may be used as a novel anticancer agent for the treatment of lung cancer in the future.

Ge W, Yin Q, Xian H
Wogonin Induced Mitochondrial Dysfunction and Endoplasmic Reticulum Stress in Human Malignant Neuroblastoma Cells Via IRE1α-Dependent Pathway.
J Mol Neurosci. 2015; 56(3):652-62 [PubMed] Related Publications
Wogonin, a flavonoid isolated from Scutellaria baicalensis Georgi, has been reported to exhibit a variety of biological effects including anti-cancer effects. It has a pro-apoptotic role in many cancer types. However, the molecular mechanisms of wogonin in treating neuroblastoma remain elusive. In the present study, two malignant neuroblastoma cell lines (SK-N-BE2 and IMR-32 cells) were treated with different doses of wogonin (0-150 μM). Wogonin showed significant cytotoxic effects in SK-N-BE2 and IMR-32 cells in a dose- and time-dependent manner. Treatment of SK-N-BE2 and IMR-32 cells with 75 μΜ wogonin for 48 h significantly promoted apoptosis, the release of cytochrome c, altered the expression of certain members of Bcl-2 family (Bcl-2, Bax and Bid), and increased the activation of caspase-3, caspase-8, caspase-9, and PARP-1, which demonstrated that the cytotoxic effect of wogonin in SK-N-BE2 and IMR-32 cells is mediated by mitochondrial dysfunction. Moreover, wogonin induced the expression of endoplasmic reticulum (ER) stress-related proteins (GRP78/Bip and GRP94/gp96) and activation of caspase-12 and caspase-4 in SK-N-BE2 and IMR-32 cells. In addition, wogonin increase the expression of IRE1α and TRAF2, and phosphorylation of ASK1 and JNK in SK-N-BE2 and IMR-32 cells. Knockdown of IRE1α by siRNA not only markedly inhibited wogonin-induced up-regulation of IRE1α and TRAF2, and phosphorylation of ASK1 and JNK but also reduced wogonin-induced cytotoxic effects and mitochondrial dysfunction in SK-N-BE2 and IMR-32 cells. These results indicated that wogonin could induce apoptosis, mitochondrial dysfunction, and ER stress in SK-N-BE2 and IMR-32 cells by modulating IRE1α-dependent pathway.

Yang HJ, Wang M, Wang L, et al.
NF-κB regulates caspase-4 expression and sensitizes neuroblastoma cells to Fas-induced apoptosis.
PLoS One. 2015; 10(2):e0117953 [PubMed] Free Access to Full Article Related Publications
Found in neurons and neuroblastoma cells, Fas-induced apoptosis and accompanied activation of NF-κB signaling were thought to be associated with neurodegenerative diseases. However, the detailed functions of NF-κB activation in Fas killing and the effect of NF-κB activation on its downstream events remain unclear. Here, we demonstrated that agonistic Fas antibody induces cell death in a dose-dependent way and NF-κB signaling is activated as well, in neuroblastoma cells SH-EP1. Unexpectedly, NF-κB activation was shown to be pro-apoptotic, as suggested by the reduction of Fas-induced cell death with either a dominant negative form of IκBα (DN-IκBα) or an IκB kinase-specific inhibitor. To our interest, when analyzing downstream events of NF-κB signaling, we found that DN-IκBα only suppressed the expression of caspase-4, but not other caspases. Vice versa, enhancement of NF-κB activity by p65 (RelA) overexpression increased the expression of caspase-4 at both mRNA and protein levels. More directly, results from dual luciferase reporter assay demonstrated the regulation of caspase-4 promoter activity by NF-κB. When caspase-4 activity was blocked by its dominant negative (DN) form, Fas-induced cell death was substantially reduced. Consistently, the cleavage of PARP and caspase-3 induced by Fas was also reduced. In contrast, the cleavage of caspase-8 remained unaffected in caspase-4 DN cells, although caspase-8 inhibitor could rescue Fas-induced cell death. Collectively, these data suggest that caspase-4 activity is required for Fas-induced cell apoptosis and caspase-4 may act upstream of PARP and caspase-3 and downstream of caspase-8. Overall, we demonstrate that NF-κB can mediate Fas-induced apoptosis through caspase-4 protease, indicating that caspase-4 is a new mediator of NF-κB pro-apoptotic pathway in neuroblastoma cells.

Kuo KL, Ho IL, Shi CS, et al.
MLN4924, a novel protein neddylation inhibitor, suppresses proliferation and migration of human urothelial carcinoma: In vitro and in vivo studies.
Cancer Lett. 2015; 363(2):127-36 [PubMed] Related Publications
MLN4924, a small molecule inhibitor of NEDD8 activating enzyme (NAE), has been reported to elicit an anti-tumor effect on various malignancies. In this study, we investigated the anti-tumor effect of MLN4924 in human urothelial carcinoma (UC) in vitro and in vivo by using three human UC cell lines of various grading (T24, NTUB1 and RT4). The impact of MLN4924 on UC cells was determined by measuring viability (MTT), proliferation (BrdU incorporation), cell cycle progression (flow cytometry with propidium iodide staining) and apoptosis (flow cytometry with annexin V-FITC labeling). The cell cycle regulatory molecules, apoptosis-related molecules, and cell stress-related proteins were examined by Western blotting. The influence of tumor cell migration and invasion was analyzed by Transwell and wound healing assays. We also evaluated the effects of MLN4924 on tumor growth by a SCID xenograft mouse model. The data show that MLN4924 induced dose-dependent cytotoxicity, anti-proliferation, anti-migration, anti-invasion and apoptosis in human UC cells, accompanied by activations of Bad, phospho-histone H2A.X, caspase-3, 7 and PARP, decreased level of phospho-Bcl2, and caused cell cycle retardation at the G2M phase. Moreover, MLN4924 activated endoplasmic reticulum stress-related molecules (caspase-4, phospho-eIF2α, ATF-4 and CHOP) and other stress responses (JNK and c-Jun activations). Finally, we confirmed MLN4924 inhibited tumor growth in a UC xenograft mouse model with minimal general toxicity. We concluded that MLN4924 induces apoptosis and cell cycle arrest, as well as activation of cell stress responses in human UC. These findings imply MLN4924 provides a novel strategy for the treatment of UC.

Kaku Y, Tsuchiya A, Kanno T, et al.
The newly synthesized anticancer drug HUHS1015 is useful for treatment of human gastric cancer.
Cancer Chemother Pharmacol. 2015; 75(3):527-35 [PubMed] Free Access to Full Article Related Publications
Naftopidil is clinically for treatment of benign prostate hyperplasia, and emerging evidence has pointed to its anticancer effect. To obtain the anticancer drug with the potential greater than that of naftopidil, we have newly synthesized the naftopidil analogue HUHS1015. The present study investigated the mechanism underlying HUHS1015-induced apoptosis of human gastric cancer cells and assessed the possibility for clinical use as an innovative anticancer drug. HUHS1015 reduced cell viability for MKN28 human well-differentiated gastric adenocarcinoma cell line and MKN45 human poorly differentiated gastric adenocarcinoma cell line in a concentration (0.3-100 μM)-dependent manner more effectively than cisplatin, a chemo-drug widely used. In the flow cytometry using propidium iodide (PI) and annexin V, HUHS1015 significantly increased the population of PI-positive and annexin V-negative cells, corresponding to primary necrosis and that of PI-positive and annexin V-positive cells, corresponding to late apoptosis/secondary necrosis, both in the two cell types. HUHS1015 significantly activated caspase-3, caspase-4, and caspase-8 in MKN45 cells, while no obvious caspase activation was found in MKN28 cells. HUHS1015 upregulated expression of the tumor necrosis factor α (TNFα) mRNA and protein in MKN45 cells, allowing activation of caspase-8 through TNF receptor and the effector caspase-3. HUHS1015 clearly inhibited tumor growth in mice inoculated with MKN45 cells, with the survival rate higher than that for the anticancer drugs cisplatin, paclitaxel, and irinotecan. The results of the present study show that HUHS1015 induces caspase-independent and caspase-dependent apoptosis of MKN28 and MKN45 human gastric cancer cells, respectively, and effectively suppresses MKN45 cell proliferation.

Sollberger G, Strittmatter GE, Grossi S, et al.
Caspase-1 activity is required for UVB-induced apoptosis of human keratinocytes.
J Invest Dermatol. 2015; 135(5):1395-404 [PubMed] Related Publications
Caspase-1 has a crucial role in innate immunity as the protease activates the proinflammatory cytokine prointerleukin(IL)-1β. Furthermore, caspase-1 induces pyroptosis, a lytic form of cell death that supports inflammation. Activation of caspase-1 occurs in multi-protein complexes termed inflammasomes, which assemble upon sensing of stress signals. In the skin and in skin-derived keratinocytes, UVB irradiation induces inflammasome-dependent IL-1 secretion and sunburn. Here we present evidence that caspase-1 and caspase-4 are required for UVB-induced apoptosis. In UVB-irradiated human primary keratinocytes, apoptosis occurs significantly later than inflammasome activation but depends on caspase-1 activity. However, it proceeds independently of inflammasome activation. By a proteomics approach, we identified the antiapoptotic Bap31 as a putative caspase-1 substrate. Caspase-1-dependent apoptosis is possibly a recent process in evolution as it was not detected in mice. These results suggest a protective role of caspase-1 in keratinocytes during UVB-induced skin cancer development through the induction of apoptosis.

Meng P, Yoshida H, Tanji K, et al.
Carnosic acid attenuates apoptosis induced by amyloid-β 1-42 or 1-43 in SH-SY5Y human neuroblastoma cells.
Neurosci Res. 2015; 94:1-9 [PubMed] Related Publications
Amyloid-beta (Aβ) peptides, Aβ 1-42 (Aβ42) and Aβ43 in particular, cause neurotoxicity and cell death in the brain of Alzheimer's disease (AD) at higher concentrations. Carnosic acid (CA), a phenolic diterpene compound in the labiate herbs rosemary and sage, serves as an activator for neuroprotective and neurotrophic functions in brain cells. We investigated the effect of CA on apoptosis induced by Aβ42 or Aβ43 in cultured SH-SY5Y human neuroblastoma cells. Treatment of the cells with Aβ42 or Aβ43 (monomer, 10 μM each) induced apoptosis, which was confirmed by the cleavage of poly-(ADP-ribose) polymerase (PARP) and apoptosis-inducing factor (AIF). Concurrently, the Aβ treatment induced the activation of caspase (Casp) cascades including an effector Casp (Casp3) and initiator Casps (Casp4, Casp8 and Casp9). Pretreatment of the cells with CA (10 μM) partially attenuated the apoptosis induced by Aβ42 or Aβ43. CA pretreatment also reduced the cellular oligomers of Aβ42 and Aβ43. These results suggest that CA suppressed the activation of Casp cascades by reducing the intracellular oligomerization of exogenous Aβ42/43 monomer. The ingestion of an adequate amount of CA may have a potential in the prevention of Aβ-mediated diseases, particularly AD.

Hoskins JW, Jia J, Flandez M, et al.
Transcriptome analysis of pancreatic cancer reveals a tumor suppressor function for HNF1A.
Carcinogenesis. 2014; 35(12):2670-8 [PubMed] Free Access to Full Article Related Publications
Pancreatic ductal adenocarcinoma (PDAC) is driven by the accumulation of somatic mutations, epigenetic modifications and changes in the micro-environment. New approaches to investigating disruptions of gene expression networks promise to uncover key regulators and pathways in carcinogenesis. We performed messenger RNA-sequencing in pancreatic normal (n = 10) and tumor (n = 8) derived tissue samples, as well as in pancreatic cancer cell lines (n = 9), to determine differential gene expression (DE) patterns. Sub-network enrichment analyses identified HNF1A as the regulator of the most significantly and consistently dysregulated expression sub-network in pancreatic tumor tissues and cells (median P = 7.56×10(-7), median rank = 1, range = 1-25). To explore the effects of HNF1A expression in pancreatic tumor-derived cells, we generated stable HNF1A-inducible clones in two pancreatic cancer cell lines (PANC-1 and MIA PaCa-2) and observed growth inhibition (5.3-fold, P = 4.5×10(-5) for MIA PaCa-2 clones; 7.2-fold, P = 2.2×10(-5) for PANC-1 clones), and a G0/G1 cell cycle arrest and apoptosis upon induction. These effects correlated with HNF1A-induced down-regulation of 51 of 84 cell cycle genes (e.g. E2F1, CDK2, CDK4, MCM2/3/4/5, SKP2 and CCND1), decreased expression of anti-apoptotic genes (e.g. BIRC2/5/6 and AKT) and increased expression of pro-apoptotic genes (e.g. CASP4/9/10 and APAF1). In light of the established role of HNF1A in the regulation of pancreatic development and homeostasis, our data suggest that it also functions as an important tumor suppressor in the pancreas.

Hsia TC, Yu CC, Hsu SC, et al.
Cantharidin induces apoptosis of H460 human lung cancer cells through mitochondria-dependent pathways.
Int J Oncol. 2014; 45(1):245-54 [PubMed] Related Publications
Lung cancer is one of the leading causes of death in cancer-related diseases. Cantharidin (CTD) is one of the components of natural mylabris (Mylabris phalerata Pallas). Numerous studies have shown that CTD induced cytotoxic effects on cancer cells. However, there is no report to demonstrate that CTD induced apoptosis in human lung cancer cells. Herein, we investigated the effect of CTD on the cell death via the induction of apoptosis in H460 human lung cancer cells. Flow cytometry assay was used for examining the percentage of cell viability, sub-G1 phase of the cell cycle, reactive oxygen species (ROS) and Ca²⁺ productions and the levels of mitochondrial membrane potential (∆Ψm). Annexin V/PI staining and DNA gel electrophoresis were also used for examining cell apoptosis. Western blot analysis was used to examine the changes of apoptosis associated protein expression and confocal microscopy for examining the translocation apoptosis associated protein. Results indicated that CTD significantly induced cell morphological changes and decreased the percentage of viable H460 cells. CTD induced apoptosis based on the occurrence of sub-G1 phase and DNA fragmentation. We found that CTD increased gene expression (mRNA) of caspase-3 and -8. Moreover, CTD increased ROS and Ca2+ production and decreased the levels of ∆Ψm. Western blot analysis results showed that CTD increased the expression of cleavage caspase-3 and -8, cytochrome c, Bax and AIF but inhibited the levels of Bcl-xL. CTD promoted ER stress associated protein expression such as GRP78, IRE1α, IRE1β, ATF6α and caspase-4 and it also promoted the expression of calpain 2 and XBP-1, but inhibited calpain 1 that is associated with apoptosis pathways. Based on those observations, we suggest that CTD may be used as a novel anticancer agent for the treatment of lung cancer in the future.

Hui KF, Chiang AK
Combination of proteasome and class I HDAC inhibitors induces apoptosis of NPC cells through an HDAC6-independent ER stress-induced mechanism.
Int J Cancer. 2014; 135(12):2950-61 [PubMed] Related Publications
The current paradigm stipulates that inhibition of histone deacetylase (HDAC) 6 is essential for the combinatorial effect of proteasome and HDAC inhibitors for the treatment of cancers. Our study aims to investigate the effect of combining different class I HDAC inhibitors (without HDAC6 action) with a proteasome inhibitor on apoptosis of nasopharyngeal carcinoma (NPC). We found that combination of a proteasome inhibitor, bortezomib, and several class I HDAC inhibitors, including MS-275, apicidin and romidepsin, potently induced killing of NPC cells both in vitro and in vivo. Among the drug pairs, combination of bortezomib and romidepsin (bort/romidepsin) was the most potent and could induce apoptosis at low nanomolar concentrations. The apoptosis of NPC cells was reactive oxygen species (ROS)- and caspase-dependent but was independent of HDAC6 inhibition. Of note, bort/romidepsin might directly suppress the formation of aggresome through the downregulation of c-myc. In addition, two markers of endoplasmic reticulum (ER) stress-induced apoptosis, ATF-4 and CHOP/GADD153, were upregulated, whereas a specific inhibitor of caspase-4 (an initiator of ER stress-induced apoptosis) could suppress the apoptosis. When ROS level in the NPC cells was reduced to the untreated level, ER stress-induced caspase activation was abrogated. Collectively, our data demonstrate a model of synergism between proteasome and class I HDAC inhibitors in the induction of ROS-dependent ER stress-induced apoptosis of NPC cells, independent of HDAC6 inhibition, and provide the rationale to combine the more specific and potent class I HDAC inhibitors with proteasome inhibitors for the treatment of cancers.

Silva JC, Ferreira-Strixino J, Fontana LC, et al.
Apoptosis-associated genes related to photodynamic therapy in breast carcinomas.
Lasers Med Sci. 2014; 29(4):1429-36 [PubMed] Related Publications
The aim of this study was to find the apoptosis molecular markers involved in the cell death that might be related to photodynamic therapy (PDT) mechanisms in breast cancer. The mammary tumors were induced in 25 Sprague-Dawley female rats by a single, oral gavage of 7,12-dimethylbenz(a)anthracene (DMBA; 70 mg/kg body weight). Animals were divided into four groups: G1 (normal, without DMBA), G2 (control, without PDT treatment), G3 (euthanized 48 h after PDT), and G4 (euthanized 24 h after PDT). For PDT experiments, the photosensitizer used was Photodithazine, and 100 J/cm of light at a fluence rate of 100 mW/cm was delivered to treat lesions. A sample of each animal was investigated by quantitative real-time PCR using Rat Apoptosis RT2 Profiler™ PCR Array platform. The results showed 20 genes with differential expression between PDT and control groups. A significant upregulation was observed for pro-apoptotic genes CASP4, CASP12, CIDEA, GADD45A, and FAS and downregulation of anti-apoptotic genes MAPK8IP1, TNFRSF11B, and NAIP2 in PDT-treated tumors. These results indicate that these genes are more directly involved in cell apoptosis induced by PDT.

Johnson GG, White MC, Wu JH, et al.
The deadly connection between endoplasmic reticulum, Ca2+, protein synthesis, and the endoplasmic reticulum stress response in malignant glioma cells.
Neuro Oncol. 2014; 16(8):1086-99 [PubMed] Free Access to Full Article Related Publications
BACKGROUND: The endoplasmic reticulum (ER) is involved in Ca(2+) signaling and protein processing. Accumulation of unfolded proteins following ER Ca(2+) depletion triggers the ER stress response (ERSR), which facilitates protein folding and removal of damaged proteins and can induce cell death. Unfolded proteins bind to chaperones, such as the glucose-regulated protein (GRP)78 and cause the release of GRP78-repressed proteins executing ERSR.
METHODS: Several glioma cell lines and primary astrocytes were used to analyze ERSR using standard western blots, reverse transcription-PCR, viability assays, and single cell Ca(2+) imaging.
RESULTS: ERSR induction with thapsigargin results in a more intense ERSR associated with a larger loss of ER Ca(2+), activation of ER-associated caspases (4/12) and caspase 3, and a higher rate of malignant glioma cell death than in normal glial cells. Malignant glioma cells have higher levels of protein synthesis and expression of the translocon (a component of the ribosomal complex, guiding protein entry in the ER), the activity of which is associated with the loss of ER Ca(2+). Our experiments confirm increased expression of the translocon in malignant glioma cells. In addition, blockade of the ribosome-translocon complex with agents differently affecting translocon Ca(2+) permeability causes opposite effects on ERSR deployment and death of malignant glioma cells.
CONCLUSIONS: Excessive ER Ca(2+) loss due to translocon activity appears to be responsible for the enhancement of ERSR, leading to the death of glioma cells. The results reveal a characteristic of malignant glioma cells that could be exploited to develop new therapeutic strategies to treat incurable glial malignancies.

Shen S, Zhang Y, Wang Z, et al.
Bufalin induces the interplay between apoptosis and autophagy in glioma cells through endoplasmic reticulum stress.
Int J Biol Sci. 2014; 10(2):212-24 [PubMed] Free Access to Full Article Related Publications
Malignant gliomas are common primary tumors of the central nervous system. The prognosis of patients with malignant glioma is poor in spite of current intensive therapy and thus novel therapeutic modalities are necessary. Bufalin is the major component of Chan-Su (a traditional Chinese medicine) extracts from the venom of Bufo gargarizan. In this study, we evaluated the growth inhibitory effect of bufalin on glioma cells and explored the underlying molecular mechanisms. Our results showed that bufalin inhibited the growth of glioma cells significantly. Mechanistic studies demonstrated that bufalin induced apoptosis through mitochondrial apoptotic pathway. In addition, bufalin was also found to induce ER stress-mediated apoptosis, which was supported by the up- regulation of ER stress markers, CHOP and GRP78, and augmented phosphorylation of PERK and eIF2α as well as cleavage of caspase-4. Downregulation of CHOP using siCHOP RNA attenuated bufalin-induced apoptosis, further confirming the role of ER stress response in mediating bufalin-induced apoptosis. Evidence of bufalin-induced autophagy included formation of the acidic vesicular organelles, increase of autophagolysosomes and LC3-II accumulation. Further experiments showed that the mechanism of bufalin-induced autophagy associated with ATP deleption involved an increase in the active form of AMPK, decreased phosphorylation levels of mTOR and its downstream targets 4EBP1 and p70S6K1. Furthermore, TUDC and silencing of eIF2α or CHOP partially blocked bufalin-induced accumulation of LC3-II, which indicated that ER stress preceded bufalin-induced autophagy and PERK/eIF2α/CHOP signaling pathway played a major part in the process. Blockage of autophagy increased expression of ER stress associated proteins and the ratio of apoptosis, indicating that autophagy played a cytoprotective role in bufalin induced ER stress and cell death. In conclusion, bufalin inhibits glioma cell growth and induces interplay between apoptosis and autophagy through endoplasmic reticulum stress. It will provide molecular bases for developing bufalin into a drug candidate for the treatment of maglinant glioma.

Nakajima A, Tsuji M, Inagaki M, et al.
Neuroprotective effects of propofol on ER stress-mediated apoptosis in neuroblastoma SH-SY5Y cells.
Eur J Pharmacol. 2014; 725:47-54 [PubMed] Related Publications
Anesthetic treatment has been associated with widespread apoptotic neurodegeneration in the neonatal rodent brain. It has recently been suggested that propofol, a short-acting intravenous anesthetic agent, may have a potential as a neuroprotective agent. An apoptotic pathway mediated through endoplasmic reticulum (ER) stress has been attracting attention. ER stress is associated with accumulation of unfolded or misfolded proteins in ER, and ER stress-induced apoptosis is implicated in a wide range of diseases, including ischemia/reperfusion injury, neurodegeneration, and diabetes. We investigated whether thapsigargin-induced ER stress is prevented by propofol in human neuroblastoma SH-SY5Y cells. SH-SY5Y cells were pretreated with various concentrations of propofol (1-10 μM) for 3h before co-treatment with 0.5 μM thapsigargin and propofol for 20 h. Levels of ssDNA, specific evidence of apoptosis, and biomarkers of ER stress (mRNA expression of Chop and sXbp-1) were determined. We also assayed calpain and caspase-4 activities and intracellular Ca(2+) ([Ca(2+)]i) levels. Thapsigargin-induced increases in ssDNA levels, expressions of ER stress biomarkers, activities of caspase-4 and calpain, and level of [Ca(2+)]i were suppressed by co-incubation with propofol. Our data indicate the possibility that propofol inhibits the Ca(2+) release from ER at clinically employed dose levels. These results demonstrate that propofol suppresses the ER stress-induced apoptosis in this cell system, and may have the neuroprotective potency. It may also be a promising agent for preventing damage from cerebral ischemia or edema.

Wu LF, Guo YT, Zhang QH, et al.
Enhanced antitumor effects of adenoviral-mediated siRNA against GRP78 gene on adenosine-induced apoptosis in human hepatoma HepG2 cells.
Int J Mol Sci. 2014; 15(1):525-44 [PubMed] Free Access to Full Article Related Publications
Our previous studies show that adenosine-induced apoptosis is involved in endoplasmic reticulum stress in HepG2 cells. In this study, we have investigated whether knockdown of GRP78 by short hairpin RNA (shRNA) increases the cytotoxic effects of adenosine in HepG2 cells. The adenovirus vector-delivered shRNA targeting GRP78 (Ad-shGRP78) was constructed and transfected into HepG2 cells. RT-PCR assay was used to determine RNA interference efficiency. Effects of knockdown of GRP78 on adenosine-induced cell viabilities, cell-cycle distribution and apoptosis, as well as relative protein expressions were determined by flow cytometry and/or Western blot analysis. The intracellular Ca2+ concentration was detected by laser scanning confocal microscope. Mitochondrial membrane potential (ΔΨm) was measured by a fluorospectrophotometer. The results revealed that GRP78 mRNA was significantly downregulated by Ad-shGRP78 transfection. Knockdown of GRP78 enhanced HepG2 cell sensitivity to adenosine by modulating G0/G1 arrest and stimulating Bax, Bak, m-calpain, caspase-4 and CHOP protein levels. Knockdown of GRP78 worsened cytosolic Ca2+ overload and ΔΨm loss. Knockdown of caspase-4 by shRNA decreased caspase-3 mRNA expression and cell apoptosis. These findings indicate that GRP 78 plays a protective role in ER stress-induced apoptosis and show that the combination of chemotherapy drug and RNA interference adenoviruses provides a new treatment strategy against malignant tumors.

Chow SE, Kao CH, Liu YT, et al.
Resveratrol induced ER expansion and ER caspase-mediated apoptosis in human nasopharyngeal carcinoma cells.
Apoptosis. 2014; 19(3):527-41 [PubMed] Related Publications
Autophagy and endoplasmic reticulum (ER) stress response is important for cancer cells to maintain malignancy and resistance to therapy. trans-Resveratrol (RSV), a non-flavonoid agent, has been shown to induce apoptosis in human nasopharyngeal carcinoma (NPC) cells. In this study, the involvements of tumor-specific ER stress and autophagy in the RSV-mediated apoptosis were investigated. In addition to traditional autophagosomes, the images of transmission electron microscopy (TEM) indicated that RSV markedly induced larger, crescent-shaped vacuoles with single-layered membranes whose the expanded cisternae contains multi-lamellar membrane structures. Prolonged exposure to RSV induced a massive accumulation of ER expansion. Using an EGFP-LC3B transfection and confocal laser microscopy approach, we found RSV-induced EGFP-LC3 puncta co-localized with ER-tracker red dye, implicating the involvement of LC3II in ER expansion. The proapoptotic effect of RSV was enhanced after suppression of autophagy by ATG7 siRNA or blocking the autophagic flux by bafilomycin A1, but that was not changed after targeted silence of IRE1 or CHOP by siRNA. Using caspase inhibitors, we demonstrated the upregulation of caspase-12 (casp12) and the activation of casp4 were associated with the proapoptotic induction of RSV through the caspase-9/caspase-3 pathway. Intriguingly, siRNA knockdown of casp12, but not caspase-4, decreased the susceptibility of the NPC cells to RSV-mediated apoptosis. Further, we showed that RSV dose-dependently increased the ceramide accumulation as assessed by LC-MS/MS system. Using serine palmitoyltransferase (SPT, a key enzyme of de novo ceramide biosynthesis) inhibitors (L-cycloserine and myriocin), we found the increased ceramide accumulation was strongly correlated with the proapoptotic potential of RSV. This study revealed the ER expansion and upregulation of ER casp12 together may indicate profound biological effects of RSV and contributed to NPC cell death. Targeting the different status of ER stress may provide a possible strategy for cancer treatments.

Chen Y, Tsai YH, Tseng SH
RECK regulated endoplasmic reticulum stress response and enhanced cisplatin-induced cell death in neuroblastoma cells.
Surgery. 2013; 154(5):968-79 [PubMed] Related Publications
BACKGROUND: Reversion-inducing-cysteine-rich protein with Kazal motifs (RECK) is critical for the invasiveness and metastasis of tumor cells; however, its role in regulating the endoplasmic reticulum (ER) stress response remains unclear. In this study we investigated the protein that interacts with RECK and the effects of RECK overexpression on the ER stress response and on cisplatin-induced cell death in neuroblastoma cells.
METHODS: Full-length RECK (FL-RECK) or a C-terminus-deleted mutant of RECK (del-C-RECK) was transfected into neuroblastoma cells. An immunoprecipitation (IP) assay and liquid chromatography with tandem mass spectrometry (LC-MS/MS) analysis were used to identify the RECK-interacting proteins. The interaction between RECK and these proteins was confirmed using co-IP and an immunofluorescence assay. Phosphorylation of double-stranded, RNA-activated protein kinase-like, ER-localized eukaryotic initiation factor-2α (eIF-2α) kinase (PERK) and eIF-2α, and expression of ER stress-related apoptotic factors were studied by Western blot analysis.
RESULTS: Glucose-regulated protein 78 (GRP78) was identified as the RECK-interacting protein in neuroblastoma cells, and the C-terminus region of the RECK protein was shown to interact with GRP78. Overexpression of FL-RECK, but not of del-C-RECK, increased the phosphorylation of PERK and eIF-2α in neuroblastoma cells. With cisplatin treatment, the expression of phosphorylated PERK and eIF-2α, CCAAT/enhancer-binding protein-homologous protein, Bax, and caspase-4 and -7 was higher and the cell viability was lower (P < .01) in FL-RECK-overexpressing cells than in del-C-RECK-overexpressing or vector control cells.
CONCLUSION: RECK regulated the cellular ER stress response through interaction with GRP78 and enhanced cisplatin-induced cell death in neuroblastoma cells.

Fan LM, Su J, Dong H, et al.
[Inhibition of GRP78 expression reverses cisplatin resistance in human ovarian cancer].
Zhonghua Yi Xue Za Zhi. 2013; 93(17):1341-4 [PubMed] Related Publications
OBJECTIVE: To explore the effects of GRP78 suppression on the sensitivity to cisplatin and elucidate the role and mechanism of GRP78 in ovarian cancer cisplatin resistance.
METHODS: The GRP78 siRNA expression plasmid was constructed to suppress GRP78 expression. Cell viability was detected by methyl thiazolyl tetrazolium (MTT) assay. Endoplasmic reticulum stress-related apoptosis related protein expressions were detected by reverse transcription-polymerase chain reaction (RT-PCR) and Western blot. And cell apoptosis was detected by flow cytometry.
RESULTS: The expressions of GRP78, CHOP and cleaved-caspase 4 were induced significantly by cisplatin (6 mg/L) in SKOV3 cells. And the expression of GRP78 was induced significantly by cisplatin in SKOV3/DDP cells. But the expressions of CHOP and cleaved-caspase 4 showed no significant difference. Inhibition of GRP78 expression and cisplatin combined treatment significantly increased the expressions of cleaved-caspase 4 and cleaved-caspase 3 in SKOV3/DDP cells. Inhibition of GRP78 expression reduced the cisplatin-induced up-regulations of p-Akt and p-mTOR and induced XBP1 mRNA shear expression and CHOP mRNA expression.
CONCLUSION: Inhibition of GRP78 expression reverses cisplatin resistance in SKOV3/DDP cells. The mechanism may be through the activity of Akt/mTOR signaling pathway, CHOP expression levels and caspase activity.

Vaeteewoottacharn K, Kariya R, Matsuda K, et al.
Perturbation of proteasome function by bortezomib leading to ER stress-induced apoptotic cell death in cholangiocarcinoma.
J Cancer Res Clin Oncol. 2013; 139(9):1551-62 [PubMed] Related Publications
PURPOSE: Cholangiocarcinoma (CCA) or cancer of the biliary tract is heterogeneous; however, chronic inflammatory-related features are unique in CCA. Moreover, the genes involved in proteasome functions are evidently increased in CCA. Hence, CCA might be vulnerable to endoplasmic reticulum (ER) stressors, particularly a proteasome inhibitor. Therefore, bortezomib (BTZ), a specific 26S proteasome inhibitor, was selected, and its antitumor effects against CCA were investigated.
METHODS: Liver fluke-associated CCA cell lines were used. Cell proliferation and apoptosis detection were determined by a tetrazolium-based assay, caspase detection and annexin V binding assay. The accumulations of proteasome substrates, the inductions of ER stress and unfolded protein response (UPR) proteins were demonstrated by western blot and reporter systems. The in vivo anti-proliferative effect was accessed in a subcutaneous transplantation mouse model.
RESULTS: BTZ inhibited CCA proliferation and induced caspase-dependent apoptosis, independently of the NF-κB pathway. Inhibition of protein degradation by BTZ led to the induction of UPR; induction of XBP1 splicing, ATF6 proteolysis and nuclear ATF4 as well as BiP and CHOP expressions were evident. Nevertheless, ER stress-induced UPR was overwhelming, leading to the activation of apoptosis demonstrated by proteolytic cleavages of ER-related caspase 4 and 12 as well as classical caspase 8, 9 and 3. The growth inhibitory effect of BTZ was supported by an in vivo model.
CONCLUSION: BTZ treatment could be a promising therapeutic approach for CCA treatment.

Carew JS, Espitia CM, Zhao W, et al.
Reolysin is a novel reovirus-based agent that induces endoplasmic reticular stress-mediated apoptosis in pancreatic cancer.
Cell Death Dis. 2013; 4:e728 [PubMed] Free Access to Full Article Related Publications
Activating mutation of KRas is a genetic alteration that occurs in the majority of pancreatic tumors and is therefore an ideal therapeutic target. The ability of reoviruses to preferentially replicate and induce cell death in transformed cells that express activated Ras prompted the development of a reovirus-based formulation for cancer therapy called Reolysin. We hypothesized that Reolysin exposure would trigger heavy production of viral products leading to endoplasmic reticular (ER) stress-mediated apoptosis. Here, we report that Reolysin treatment stimulated selective reovirus replication and decreased cell viability in KRas-transformed immortalized human pancreatic duct epithelial cells and pancreatic cancer cell lines. These effects were associated with increased expression of ER stress-related genes, ER swelling, cleavage of caspase-4, and splicing of XBP-1. Treatment with ER stress stimuli including tunicamycin, brefeldin A, and bortezomib (BZ) augmented the anticancer activity of Reolysin. Cotreatment with BZ and Reolysin induced the simultaneous accumulation of ubiquitinated and viral proteins, resulting in enhanced levels of ER stress and apoptosis in both in vitro and in vivo models of pancreatic cancer. Our collective results demonstrate that the abnormal protein accumulation induced by the combination of Reolysin and BZ promotes heightened ER stress and apoptosis in pancreatic cancer cells and provides the rationale for a phase I clinical trial further investigating the safety and efficacy of this novel strategy.

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