Transglutaminase 2 Promotes Autophagy by LC3 Induction through p53 Depletion in Cancer Cell

Transglutaminase 2 (TGase 2) plays a key role in p53 regulation, depleting p53 tumor suppressor through autophagy in renal cell carcinoma. We found that microtubule-associated protein 1A/1B-light chain 3 (LC3), a hallmark of autophagy, were tightly associated with the level of TGase 2 in cancer cells. TGase 2 overexpression increased LC3 levels, and TGase 2 knockdown decreased LC3 levels in cancer cells. Transcript abundance of LC3 was inversely correlated with level of wild type p53. TGase 2 knockdown using siRNA, or TGase 2 inhibition using GK921 significantly reduced autophagy through reduction of LC3 transcription, which was followed by restoration of p53 levels in cancer cells. TGase 2 overexpression promoted the autophagy process by LC3 induction, which was correlated with p53 depletion in cancer cells. Rapamycin-resistant cancer cells also showed higher expression of LC3 compared to the rapamycin-sensitive cancer cells, which was tightly correlated with TGase 2 levels. TGase 2 knockdown or TGase 2 inhibition sensitized rapamycin-resistant cancer cells to drug treatment. In summary, TGase 2 induces drug resistance by potentiating autophagy through LC3 induction via p53 regulation in cancer.

Cancer Metabolism: a Hope for Curing Cancer

No abstract available.

Cancer Energy Metabolism: Shutting Power off Cancer Factory

In 1923, Dr. Warburg had observed that tumors acidified the Ringer solution when 13 mM glucose was added, which was identified as being due to lactate. When glucose is the only source of nutrient, it can serve for both biosynthesis and energy production. However, a series of studies revealed that the cancer cell consumes glucose for biosynthesis through fermentation, not for energy supply, under physiological conditions. Recently, a new observation was made that there is a metabolic symbiosis in which glycolytic and oxidative tumor cells mutually regulate their energy metabolism. Hypoxic cancer cells use glucose for glycolytic metabolism and release lactate which is used by oxygenated cancer cells. This study challenged the Warburg effect, because Warburg claimed that fermentation by irreversible damaging of mitochondria is a fundamental cause of cancer. However, recent studies revealed that mitochondria in cancer cell show active function of oxidative phosphorylation although TCA cycle is stalled. It was also shown that blocking cytosolic NADH production by aldehyde dehydrogenase inhibition, combined with oxidative phosphorylation inhibition, resulted in up to 80% decrease of ATP production, which resulted in a significant regression of tumor growth in the NSCLC model. This suggests a new theory that NADH production in the cytosol plays a key role of ATP production through the mitochondrial electron transport chain in cancer cells, while NADH production is mostly occupied inside mitochondria in normal cells.

Silencing of peroxiredoxin II by promoter methylation is necessary for the survival and migration of gastric cancer cells

Peroxiredoxin (Prx), a family of ubiquitous thiol peroxidases, functions as a redox signaling regulator that controls cellular Hâ‚‚Oâ‚‚ in mammalian cells and has recently received attention for being overexpressed in various cancer types. In this study, we show that Prx type II (PrxII) is rather silenced in gastric cancer cells. PrxII expression is severely downregulated in 9 out of the 28 gastric cancer cell lines. Strikingly, PrxII expression is completely lost in three cell lines, MKN28, MKN74 and SNU484. Loss of PrxII expression is due to DNA methyltransferase 1-dependent methylation at the promoter region of the PrxII gene. Restoration of PrxII expression using a retroviral system markedly reduces the colony-forming ability and migratory activity of both MKN28 and SNU484 cells by inhibiting Src kinase. Mechanistically, PrxII peroxidase activity is essential for regulating gastric cancer cell migration. Bioinformatics analysis from The Cancer Genome Atlas stomach cancer data (STAD) revealed significantly low PrxII expression in gastric cancer patients and a negative correlation between PrxII expression and methylation levels. More importantly, low PrxII expression also strongly correlates with poor survival in cancer patients. Thus our study suggests that PrxII may be the first thiol peroxidase that simultaneously regulates both survival and metastasis in gastric cancer cells with high clinical relevance.

Migration and invasion of drug-resistant lung adenocarcinoma cells are dependent on mitochondrial activity

A small proportion of cancer cells have stem-cell-like properties, are resistant to standard therapy and are associated with a poor prognosis. The metabolism of such drug-resistant cells differs from that of nearby non-resistant cells. In this study, the metabolism of drug-resistant lung adenocarcinoma cells was investigated. The expression of genes associated with oxidative phosphorylation in the mitochondrial membrane was negatively correlated with the prognosis of lung adenocarcinoma. Because the mitochondrial membrane potential (MMP) reflects the functional status of mitochondria and metastasis is the principal cause of death due to cancer, the relationship between MMP and metastasis was evaluated. Cells with a higher MMP exhibited greater migration and invasion than those with a lower MMP. Cells that survived treatment with cisplatin, a standard chemotherapeutic drug for lung adenocarcinoma, exhibited increased MMP and enhanced migration and invasion compared with parental cells. Consistent with these findings, inhibition of mitochondrial activity significantly impeded the migration and invasion of cisplatin-resistant cells. RNA-sequencing analysis indicated that the expression of mitochondrial complex genes was upregulated in cisplatin-resistant cells. These results suggested that drug-resistant cells have a greater MMP and that inhibition of mitochondrial activity could be used to prevent metastasis of drug-resistant lung adenocarcinoma cells.

Aldehyde dehydrogenase is used by cancer cells for energy metabolism

We found that non-small-cell lung cancer (NSCLC) cells express high levels of multiple aldehyde dehydrogenase (ALDH) isoforms via an informatics analysis of metabolic enzymes in NSCLC and immunohistochemical staining of NSCLC clinical tumor samples. Using a multiple reaction-monitoring mass spectrometry analysis, we found that multiple ALDH isozymes were generally abundant in NSCLC cells compared with their levels in normal IMR-90 human lung cells. As a result of the catalytic reaction mediated by ALDH, NADH is produced as a by-product from the conversion of aldehyde to carboxylic acid. We hypothesized that the NADH produced by ALDH may be a reliable energy source for ATP production in NSCLC. This study revealed that NADH production by ALDH contributes significantly to ATP production in NSCLC. Furthermore, gossypol, a pan-ALDH inhibitor, markedly reduced the level of ATP. Gossypol combined with phenformin synergistically reduced the ATP levels, which efficiently induced cell death following cell cycle arrest.

Cancer Metabolism: Strategic Diversion from Targeting Cancer Drivers to Targeting Cancer Suppliers

Drug development groups are close to discovering another pot of gold-a therapeutic target-similar to the success of imatinib (Gleevec) in the field of cancer biology. Modern molecular biology has improved cancer therapy through the identification of more pharmaceutically viable targets, and yet major problems and risks associated with late-phase cancer therapy remain. Presently, a growing number of reports have initiated a discussion about the benefits of metabolic regulation in cancers. The Warburg effect, a great discovery approximately 70 years ago, addresses the "universality" of cancer characteristics. For instance, most cancer cells prefer aerobic glycolysis instead of mitochondrial respiration. Recently, cancer metabolism has been explained not only by metabolites but also through modern molecular and chemical biological techniques. Scientists are seeking context-dependent universality among cancer types according to metabolic and enzymatic pathway signatures. This review presents current cancer metabolism studies and discusses future directions in cancer therapy targeting bio-energetics, bio-anabolism, and autophagy, emphasizing the important contribution of cancer metabolism in cancer therapy.

Novel Suppressive Effects of Ketotifen on Migration and Invasion of MDA-MB-231 and HT-1080 Cancer Cells

The high mortality rates associated with cancer reflect the metastatic spread of tumor cells from the site of their origin. Metastasis, in fact, is the cause of 90% of cancer deaths. Therefore, considerable effort is being made to inhibit metastasis. In the present study, we screened ketotifen for anti-migratory and anti-invasive activities against MDA-MB-231 breast cancer and HT-1080 fibrosarcoma cancer cells. Cancer cell migration and invasion were measured using multi-well chambers. Additionally, western blots were used to examine the effects of ketotifen on the expressions of CDC42, Rho, Rac, and matrix metalloproteinase 9 (MMP-9). The results showed that ketotifen dose-dependently suppressed the migration and invasion of MDA-MB-231 and HT-1080 cells. Ketotifen also suppressed the expressions of CDC42, Rac, and Rho, which, significantly, are involved in MDA-MB-231 and HT-1080 cancer cell migration. Moreover, ketotifen suppressed the expression and activity of MMP-9, which is involved in degradation of the extracellular matrix leading to invasion. The overall data suggested that ketotifen suppresses the migration and invasion of MDA-MB-231 and HT-1080 cancer cells via inhibition of CDC42, Rac, Rho, and MMP-9 expression.

Involvement of Transglutaminase-2 in alpha-MSH-Induced Melanogenesis in SK-MEL-2 Human Melanoma Cells

Skin hyperpigmentation is one of the most common skin disorders caused by abnormal melanogenesis. The mechanism and key factors at play are not fully understood. Previous reports have indicated that cystamine (CTM) inhibits melanin synthesis, though its molecular mechanism in melanogenesis remains unclear. In the present study, we investigated the effect of CTM on melanin production using ELISA reader and the expression of proteins involved in melanogenesis by Western blotting, and examined the involvement of transglutaminase-2 (Tgase-2) in SK-MEL-2 human melanoma cells by gene silencing. In the results, CTM dose-dependently suppressed melanin production and dendrite extension in alpha-MSH-induced melanogenesis of SK-MEL-2 human melanoma cells. CTM also suppressed alpha-MSH-induced chemotactic migration as well as the expressions of melanogenesis factors TRP-1, TRP-2 and MITF in alpha-MSH-treated SK-MEL-2 cells. Meanwhile, gene silencing of Tgase-2 suppressed dendrite extension and the expressions of TRP-1 and TRP-2 in alpha-MSH-treated SK-MEL-2 cells. Overall, these findings suggested that CTM suppresses alpha-MSH-induced melanogenesis via Tgase-2 inhibition and that therefore, Tgase-2 might be a new target in hyperpigmentation disorder therapy.

Ethacrynic Acid Inhibits Sphingosylphosphorylcholine-Induced Keratin 8 Phosphorylation and Reorganization via Transglutaminase-2 Inhibition

Sphingosylphosphorylcholine (SPC) is significantly increased in the malicious ascites of tumor patients and induces perinuclear reorganization of keratin 8 (K8) filaments in PANC-1 cells. The reorganization contributes to the viscoelasticity of metastatic cancer cells resulting in increased migration. Recently, we reported that transglutaminase-2 (Tgase-2) is involved in SPC-induced K8 phosphorylation and reorganization. However, effects of Tgase-2 inhibitors on SPC-induced K8 phosphorylation and reorganization were not clearly studied. We found that ethacrynic acid (ECA) concentration-dependently inhibited Tgase-2. Therefore, we examined the effects of ECA on SPC-induced K8 phosphorylation and reorganization. ECA concentration-dependently suppressed the SPC-induced phosphorylation and perinuclear reorganization of K8. ECA also suppressed the SPC-induced migration and invasion. SPC induced JNK activation through Tgase-2 expression and ECA suppressed the activation and expression of JNK in PANC-1 cells. These results suggested that ECA might be useful to control Tgase-2 dependent metastasis of cancer cells such as pancreatic cancer and lung cancers.

Transglutaminase 2 Expression Predicts Progression Free Survival in Non-Small Cell Lung Cancer Patients Treated with Epidermal Growth Factor Receptor Tyrosine Kinase Inhibitor

Transglutaminase 2 (TG2), a cross-linking enzyme, is involved in drug resistance and in the constitutive activation of nuclear factor kappa B (NF-kappaB). We investigated the association of non-small cell lung cancer (NSCLC) treatment efficacy with TG2 and NF-kappaB expression in 120 patients: 102 with adenocarcinoma and 18 with other histologic types. All patients underwent surgery; 88 received adjuvant chemotherapy, with 28 receiving platinum-based doublet chemotherapy as first-line treatment and 29 receiving epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitor (TKI) therapy. Patients' TG2 and NF-kappaB expression values were calculated semiquantitatively. The median TG2 value was 50 (range, 0-300) and the median NF-kappaB value was 20 (range, 0-240). Disease-free survival did not differ between the low- and high-TG2 groups. Among patients who received palliative platinum-based doublet chemotherapy, progression free survival (PFS) was longer in the low-TG2 group than in the high-TG2 group (11.0 vs. 7.0 months; P=0.330). Among those who received EGFR-TKI therapy, PFS was also longer in the low-TG2 group than in the high-TG 2 group (11.0 vs. 2.0 months; P=0.013). Similarly, in EGFR wild-type patients treated with EGFR-TKI, PFS was longer in patients with low TG2 expression (9.0 vs. 2.0 months; P=0.013). TG2 expression levels can predict PFS in patients with NSCLC treated with EGFR-TKI.

A Novel Therapeutic Target in Inflammatory Uveitis: Transglutaminase 2 Inhibitor

PURPOSE: Our goal was to investigate the effects of inhibition of transglutaminase 2 (TGase 2) on endotoxin-induced uveitis (EIU) METHODS: EIU was induced in female Lewis rats by single footpad injections of 200 microgram of lipopolysaccharide (LPS). TGase 2 inhibitors were administered intraperitoneally 30 minutes before and at the time of LPS administration. Rats were sacrificed 24 hours after injection, and the effects of the TGase 2 inhibitors were evaluated by the number of intraocular inflammatory cells present on histologic sections and by measuring the TGase 2 activity and TGase products in the aqueous humor (AqH). TGase 2 substrates were also assayed in AqH from uveitis patients. RESULTS: Clinical indications of EIU, the number of cells present on histologic sections, and TGase 2 activity in AqH increased in a time-dependent manner, peaking 24 hours after LPS injection. Inflammation in EIU was significantly reversed by treatment with TGase inhibitors. A 23-kDa cross-linked TGase substrate was identified in the AqH from EIU rats and uveitis patients. MALDI-TOF analysis showed that this substrate in uveitis patients was human Ig kappa chain C region. CONCLUSIONS: TGase 2 activity and its catalytic product were increased in the AqH of EIU rats. TGase 2 inhibition attenuated the degree of inflammation in EIU. Safe and stable TGase inhibitors may have great potential for the treatment of inflammatory uveitis.

The Effect of Transglutaminase on the Recovery of Somatosensory Evoked Potentials in the Rat Model of Spinal Cord Injury

OBJECTIVE: The authors present an investigation of the effect of transglutaminase(TG) on the recovery of somatosensory evoked potentials in the rat model of spinal cord injury. METHODS: Female Sprague-Dawley rats(280-310g) were used for this study. Rats were divided into two groups: TG treated and control. The lesion was made by transecting the right dorsal column of the thoracic spinal cord without damage to the vasculature using specially devised micro-glass pipette after laminectomy. For TG treated group, normal saline with TG were injected into the lesion site using micro-pipette through the opening of the dura. Saltatory repetitive somatosensory evoked potentials(SSEPs) recording were carried out on post-injury 6th and 12th week. RESULTS: The amplitudes of N19 were 1.28+/-1.60 microV on 6th week, 3.45+/-3.63 microV on 12th week in control group(n=10) and 1.46+/-1.75 microV on 6th week and 5.01+/-2.65 microV on 12th week in TG-treated group(n=11). Statis-tically significant recovery of SSEPs were seen in TG-treated group(p=0.003, Wilcoxon Signed Ranks Test). In TG-treated group, vacuolated degeneration around the lesion site was rarely observed on histological evaluation. CONCLUSION: This study demonstrates the possibility of long-term survival and saltatory recording of SSEPs in small animals like rats, after selective spinal cord injury. In addition, this study shows that TG is a factor facilitating the recovery of injured axon of central nervous system.

Immunohistochemical Study on the Expression of Transglutaminase 1, 2, 3 in the Rat Kidney / 대한해부학회지

Transglutaminase is an calcium dependent enzyme involved in various biological events such as cell growth and proliferation, apoptosis, fertilization, embryogenesis, and carcinogenesis. Biochemically it can be detected in many organs but no systemic in situ localization has been carried out so far. In the present study we report the immuno-histochemical localization of TG1, 2, 3 in rat kidney tissue using newly purificated polyclonal anti-goat traglutaminase 1 and anti-rabbit polyclonal transglutaminase 2 or 3 antibody. The results are as follows 1. The presence of transglutaminase 1, 2 and 3 was demonstrated in the both renal cortex and renal medulla of the rat. Although the in situ localization patterns were very similar, strength of the immunoreactivity was different; transglutaminase 1, 2, 3 in order. 2. More strong immunoreactivity for transglutaminase 1, 2, 3 were detected in the renal tubule than the renal glomerulus. 3. The strong immunoreactivity was demonstrated in the capsule, brush border of proximal convoluted tubule and collecting duct and thin limb of Henle's loop. The functional implications of these findings are presently unknown. However, based on its wide distribution in the renal tubule, certain essential role of these enzymes in maintaining the electrolytes balance may be suggested.

Immunohistochemical Study of Differential Expression of Transglutaminase 1 in the Rat / 대한해부학회지

Transglutaminase is an calcium depedent enzyme involved in various biological events such as cell growth and proliferation, apoptosis, fertilization, embryogenesis, and carcinogenesis. Biochemically it can be detected in many organs but no systemic in situ localization has been carried out so far. Here we report the immunohistochemical localization of TG1 in rat tissue using newly purificated polyclonal anti-goat traglutaminase 1 antibody. The presence of TG 1 can be demonstrated in the kidney, liver, spleen, lung, esophagus, trachea, small intestine, testis, cerebellum of the rat. The strong immunoreactivity can be demonstrated in proximal convoluted tubule and collecting duct of the kidney, central vein of the liver, esophagus of the lamina propria, epithelium and cartilage of the trachea., submucosa and Paneth cell of the small intestine, Purkinje cell of the cerebellum. Among organs, there was no relationship between the immunoreactivity and histologic similarity. The functional implications of these findings are presently unknown. However, based on its wide distribution of the tissue certain essential role of this enzyme in survival of organism may be suggested.

Expression of Transglutaminase K in Several Skin Diseases / 대한피부과학회지

To understand the role of Transglutaminase K(TGase K) in skin diseases, the expression of TGase K protein was studied in hyperkeratotic inflammatory skin diseases, and in several different kinds of benign and malignant skin tumors. Immunohistochemistry was used to detect the TGase K protein with a new anti-human TGase K polyclonal antibody. This antibody showed the same immuno- histochemical staining pattern as in previously published immunofluorescence data with the exception that the immunohistochemical stain showed a slightly weaker stain in the granular layer. However, the staining intensity was decreased in psoriasis, which is opposite for results obtained with the widely used TGase K monoclonal antibody B.C1. In other hyperkeratotic inflammatory skin diseases, like pityriasis rubra pilaria, lichen planus, and pityriasis rosea, the staining patterns were similar while the staining intensity was decreased, compared to normal epidermis. Squamous cell carcinoma showed a diffuse staining pattern in tumor cells with a small intensity increase in well differentiated tumor cells. However, in actinic keratosis and Bowen's disease, which are thought to be precursors of squamous cell carcinoma, the staining intensity was weak. The tumor cells of basal cell carcinoma, malignant melanoma, and seborrheic keratosis showed a weak staining intensity.

Isolation and characterization of brain-specific transglutaminases from rat

The relevance of transglutaminases with neural function and several disorders has been emphasized recently. Especially, many polypeptides associated with neurodegenerative diseases are suggested to be putative transglutaminase substrates such as beta amyloid protein of Alzheimer's disease, microtubule-associated proteins and neurofilaments, etc. In addition, the CAG repeated gene products with probable polyglutamine tract, putative transglutaminase substrates, were identified in several neurodegenerative disorders. However, the identity of the brain transglutaminase has not been confirmed, because of enzymic stability and low activity. In the present experiment, we have isolated brain-specific transglutaminases, designated as TGase NI and TGase NII, which are different from other types of transglutaminases in respects of molecular weights (mw. 45 kDa, 29 kDa respectively), substrate affinity, elution profile on ion-exchange chromatography, sensitivity to proteases and ethanol, and immunological properties. The enzymes were localized specifically in the brain tissues but not in the liver tissue. And neural cells such as pheochromocytoma cell, glioma cell, primary neuronal and glial cells were shown to be enriched with TGase NI and TGase NII. The possible biological roles of the enzymes were discussed not only on the aspect of crosslinking activity but also of signal transducing capacity of the enzyme in the brain.

Calpain inhibitors reduce the cornified cell envelope formation by inhibiting proteolytic processing of transglutaminase 1

Calpain I (mu-calpain) and II (m-calpain) are well known calcium-activated neutral cysteine proteases. Many reports have shown that activation of calpain is related to cataract formation, neuronal degeneration, blood clotting, ischemic injuries, muscular dystrophy and cornified cell envelope (CE) formation. Here, we report that insoluble CE formation was reduced after treatment with calpain I inhibitor (N-acetyl-leucyl-leucyl-norleucinal) on normal human epidermal keratinocytes (NHEK), whereas serine and thiol protease inhibitors had no effect on the reduction of CE. When NHEK cells were confluent, keratinocytes were treated with various concentrations (0.5 microM-0.5 mM) of calpain I inhibitor or serine and thiol protease inhibitors under calcium induced differentiation. Insoluble CE formation was reduced about 90% in the 50 microM calpain inhibitor I treated group by day 9 of culture, whereas insoluble CE was reduced only 10% in the same condition. Interestingly TGase activity was blocked by 90% in the 0.5 mM calpain inhibitor treated group within 72 h, whereas TGase activity was retained by 80% in the 0.5 mM serine protease inhibitor treated group at 7 day treatment. Therefore it can be suggested that cysteine protease calpains might be responsible for the activation of the TGase 1 enzyme to complete insoluble CE formation during epidermal differentiation.