Proton-Sensing GPCRs in Health and Disease.

The group of proton-sensing G-protein coupled receptors (GPCRs) consists of the four receptors GPR4, TDAG8 (GPR65), OGR1 (GPR68), and G2A (GPR132). These receptors are cellular sensors of acidification, a property that has been attributed to the presence of crucial histidine residues. However, the pH detection varies considerably among the group of proton-sensing GPCRs and ranges from pH of 5.5 to 7.8. While the proton-sensing GPCRs were initially considered to detect acidic cellular environments in the context of inflammation, recent observations have expanded our knowledge about their physiological and pathophysiological functions and many additional individual and unique features have been discovered that suggest a more differentiated role of these receptors in health and disease. It is known that all four receptors contribute to different aspects of tumor biology, cardiovascular physiology, and asthma. However, apart from their overlapping functions, they seem to have individual properties, and recent publications identify potential roles of individual GPCRs in mechanosensation, intestinal inflammation, oncoimmunological interactions, hematopoiesis, as well as inflammatory and neuropathic pain. Here, we put together the knowledge about the biological functions and structural features of the four proton-sensing GPCRs and discuss the biological role of each of the four receptors individually. We explore all currently known pharmacological modulators of the four receptors and highlight potential use. Finally, we point out knowledge gaps in the biological and pharmacological context of proton-sensing GPCRs that should be addressed by future studies.

A Role for Protease Activated Receptor Type 3 (PAR3) in Nociception Demonstrated Through Development of a Novel Peptide Agonist.

The protease activated receptor (PAR) family is a group of G-protein coupled receptors (GPCRs) activated by proteolytic cleavage of the extracellular domain. PARs are expressed in a variety of cell types with crucial roles in homeostasis, immune responses, inflammation, and pain. PAR3 is the least researched of the four PARs, with little known about its expression and function. We sought to better understand its potential function in the peripheral sensory nervous system. Mouse single-cell RNA sequencing data demonstrates that PAR3 is widely expressed in dorsal root ganglion (DRG) neurons. Co-expression of PAR3 mRNA with other PARs was identified in various DRG neuron subpopulations, consistent with its proposed role as a coreceptor of other PARs. We developed a lipid tethered PAR3 agonist, C660, that selectively activates PAR3 by eliciting a Ca2+ response in DRG and trigeminal neurons. In vivo, C660 induces mechanical hypersensitivity and facial grimacing in WT but not PAR3-/- mice. We characterized other nociceptive phenotypes in PAR3-/- mice and found a loss of hyperalgesic priming in response to IL-6, carrageenan, and a PAR2 agonist, suggesting that PAR3 contributes to long-lasting nociceptor plasticity in some contexts. To examine the potential role of PAR3 in regulating the activity of other PARs in sensory neurons, we administered PAR1, PAR2, and PAR4 agonists and assessed mechanical and affective pain behaviors in WT and PAR3-/- mice. We observed that the nociceptive effects of PAR1 agonists were potentiated in the absence of PAR3. Our findings suggest a complex role of PAR3 in the physiology and plasticity of nociceptors. PERSPECTIVE: We evaluated the role of PAR3, a G-protein coupled receptor, in nociception by developing a selective peptide agonist. Our findings suggest that PAR3 contributes to nociception in various contexts and plays a role in modulating the activity of other PARs.

N-Palmitoylglycine and other N-acylamides activate the lipid receptor G2A/GPR132.

The G-protein-coupled receptor GPR132, also known as G2A, is activated by 9-hydroxyoctadecadienoic acid (9-HODE) and other oxidized fatty acids. Other suggested GPR132 agonists including lysophosphatidylcholine (LPC) have not been readily reproduced. Here, we identify N-acylamides in particular N-acylglycines, as lipid activators of GPR132 with comparable activity to 9-HODE. The order-of-potency is N-palmitoylglycine > 9-HODE ≈ N-linoleoylglycine > linoleamide > N-oleoylglycine ≈ N-stereoylglycine > N-arachidonoylglycine > N-docosehexanoylglycine. Physiological concentrations of N-acylglycines in tissue are sufficient to activate GPR132. N-linoleoylglycine and 9-HODE also activate rat and mouse GPR132, despite limited sequence conservation to human. We describe pharmacological tools for GPR132, identified through drug screening. SKF-95667 is a novel GPR132 agonist. SB-583831 and SB-583355 are peptidomimetic molecules containing core amino acids (glycine and phenylalanine, respectively), and structurally related to previously described ligands. A telmisartan analog, GSK1820795A, antagonizes the actions of N-acylamides at GPR132. The synthetic cannabinoid CP-55 940 also activates GPR132. Molecular docking to a homology model suggested a site for lipid binding, predicting the acyl side-chain to extend into the membrane bilayer between TM4 and TM5 of GPR132. Small-molecule ligands are envisaged to occupy a "classical" site encapsulated in the 7TM bundle. Structure-directed mutagenesis indicates a critical role for arginine at position 203 in transmembrane domain 5 to mediate GPR132 activation by N-acylamides. Our data suggest distinct modes of binding for small-molecule and lipid agonists to the GPR132 receptor. Antagonists, such as those described here, will be vital to understand the physiological role of this long-studied target.

Imipridone ONC212 activates orphan G protein-coupled receptor GPR132 and integrated stress response in acute myeloid leukemia.

Imipridones constitute a novel class of antitumor agents. Here, we report that a second-generation imipridone, ONC212, possesses highly increased antitumor activity compared to the first-generation compound ONC201. In vitro studies using human acute myeloid leukemia (AML) cell lines, primary AML, and normal bone marrow (BM) samples demonstrate that ONC212 exerts prominent apoptogenic effects in AML, but not in normal BM cells, suggesting potential clinical utility. Imipridones putatively engage G protein-coupled receptors (GPCRs) and/or trigger an integrated stress response in hematopoietic tumor cells. Comprehensive GPCR screening identified ONC212 as activator of an orphan GPCR GPR132 and Gαq signaling, which functions as a tumor suppressor. Heterozygous knock-out of GPR132 decreased the antileukemic effects of ONC212. ONC212 induced apoptogenic effects through the induction of an integrated stress response, and reduced MCL-1 expression, a known resistance factor for BCL-2 inhibition by ABT-199. Oral administration of ONC212 inhibited AML growth in vivo and improved overall survival in xenografted mice. Moreover, ONC212 abrogated the engraftment capacity of patient-derived AML cells in an NSG PDX model, suggesting potential eradication of AML initiating cells, and was highly synergistic in combination with ABT-199. Collectively, our results suggest ONC212 as a novel therapeutic agent for AML.

Iron chelator-induced up-regulation of Ndrg1 inhibits proliferation and EMT process by targeting Wnt/ß-catenin pathway in colon cancer cells.

Di-2-pyridylketone-4,4-dimethyl-3-thiosemicarbazone (Dp44mT), as the novel iron chelator, has been reported to inhibit the tumorigenesis and progression of various cancer cells. However, whether Dp44mT has anticancer effects in colon cancer cells is still unknown. Here, we investigated the antitumor action of Dp44mT in colon cancer and its underlying mechanisms, and the connections between Dp44mT and N-myc downstream-regulated genes 1(Ndrg1). We used cell viability, migration and invasion assay, flow cytometry, western blot and qRT-PCR to examine the anticancer effects of Dp44mT and Ndrg1. We found that Dp44mT suppressed cell viability, migration, invasion and induced apoptosis of colon cancer cells and over-expression of Ndrg1 also suppressed cell viability, migration, invasion and induced apoptosis of colon cancer cells. Dp44mT attenuated the TGF-ß1-induced EMT in colon cancer cells, and Dp44mT could up-regulate Ndrg1 expression level. Overexpression of Ndrg1 attenuates the TGF-ß1-induced EMT, Dp44mT and Ndrg1 suppressed EMT through activation of Wnt/ß catenin signaling pathway. In conclusion, our data demonstrated that Dp44mT/Ndrg1 have effective anticancer capability in colon cancer cells and that may represent a promising treatment strategy for human colon cancer.

Modulation of the Allosteric Communication between the Polo-Box Domain and the Catalytic Domain in Plk1 by Small Compounds.

The Polo-like kinases (Plks) are an evolutionary conserved family of Ser/Thr protein kinases that possess, in addition to the classical kinase domain at the N-terminus, a C-terminal polo-box domain (PBD) that binds to phosphorylated proteins and modulates the kinase activity and its localization. Plk1, which regulates the formation of the mitotic spindle, has emerged as a validated drug target for the treatment of cancer, because it is required for numerous types of cancer cells but not for the cell division in noncancer cells. Here, we employed chemical biology methods to investigate the allosteric communication between the PBD and the catalytic domain of Plk1. We identified small compounds that bind to the catalytic domain and inhibit or enhance the interaction of Plk1 with the phosphorylated peptide PoloBoxtide in vitro. In cells, two new allosteric Plk1 inhibitors affected the proliferation of cancer cells in culture and the cell cycle but had distinct phenotypic effects on spindle formation. Both compounds inhibited Plk1 signaling, indicating that they specifically act on Plk1 in cultured cells.

E. coli Nissle 1917-Derived Minicells for Targeted Delivery of Chemotherapeutic Drug to Hypoxic Regions for Cancer Therapy.

Purpose: Systemic administration of free chemotherapeutic drugs leads to severe toxic effects, and physiological characteristics of solid tumors restrain the drugs from reaching the hypoxic regions. E. coli Nissle 1917 (EcN) has been known to penetrate the barrier and proliferate in the interface between the viable and necrotic regions of tumors. This study aimed to fabricate a nanoscale minicell via genetic engineering of EcN for targeted delivery of chemotherapeutic drugs to the hypoxic regions of tumors for cancer therapy. Methods: A large number of minicells were produced by knocking out the minCD gene and enhancing the minE expression in EcN. Then, a pH (low) insertion peptide (pHLIP) was displayed on the membrane surface through protein display technology to endow the cells with the ability to target the acidic microenvironments of tumors. The acidic-microenvironment targeting ability and therapeutic effect of the engineered minicells with chemotherapeutic drugs was thoroughly evaluated by using breast cancer cells and an orthotopic model of breast tumor. Results: The EcN-derived minicells displaying pHLIP could be directly extracted from the fermentation broth and used for delivering chemotherapeutic drugs without any further modification. Targeting of doxorubicin (DOX)-loaded minicells to cancer cells via pHLIP resulted in rapid internalization and drug release in acidic media. Importantly, the pHLIP-mosaic minicells successfully invaded the necrotic and hypoxic regions of orthotopic breast cancers where free chemotherapeutic drugs could never get to because of vascular insufficiency and high interstitial fluid pressure. This invasion resulted in significant regression of an orthotopic breast tumor in a mouse model, while no seriously pathogenic effects were observed during the animal experiments. Conclusions: This study provides a novel strategy for the fabrication of tumor-targeting carriers via genetic engineering based on biomaterials with the ability to penetrate hypoxic regions of tumors, high biocompatibility and low toxicity.

GADD45a Regulates Olaquindox-Induced DNA Damage and S-Phase Arrest in Human Hepatoma G2 Cells via JNK/p38 Pathways.

Olaquindox, a quinoxaline 1,4-dioxide derivative, is widely used as a feed additive in many countries. The potential genotoxicity of olaquindox, hence, is of concern. However, the proper mechanism of toxicity was unclear. The aim of the present study was to investigate the effect of growth arrest and DNA damage 45 alpha (GADD45a) on olaquindox-induced DNA damage and cell cycle arrest in HepG2 cells. The results showed that olaquindox could induce reactive oxygen species (ROS)-mediated DNA damage and S-phase arrest, where increases of GADD45a, cyclin A, Cdk 2, p21 and p53 protein expression, decrease of cyclin D1 and the activation of phosphorylation-c-Jun N-terminal kinases (p-JNK), phosphorylation-p38 (p-p38) and phosphorylation-extracellular signal-regulated kinases (p-ERK) were involved. However, GADD45a knockdown cells treated with olaquindox could significantly decrease cell viability, exacerbate DNA damage and increase S-phase arrest, associated with the marked activation of p-JNK, p-p38, but not p-ERK. Furthermore, SP600125 and SB203580 aggravated olaquindox-induced DNA damage and S-phase arrest, suppressed the expression of GADD45a. Taken together, these findings revealed that GADD45a played a protective role in olaquindox treatment and JNK/p38 pathways may partly contribute to GADD45a regulated olaquindox-induced DNA damage and S-phase arrest. Our findings increase the understanding on the molecular mechanisms of olaquindox.

c-Rel Regulates Inscuteable Gene Expression during Mouse Embryonic Stem Cell Differentiation.

Inscuteable (Insc) regulates cell fate decisions in several types of stem cells. Although it is recognized that the expression levels of mouse INSC govern the balance between symmetric and asymmetric stem cell division, regulation of mouse Insc gene expression remains poorly understood. Here, we showed that mouse Insc expression transiently increases at an early stage of differentiation, when mouse embryonic stem (mES) cells differentiate into bipotent mesendoderm capable of producing both endoderm and mesoderm in defined culture conditions. We identified the minimum transcriptional regulatory element (354 bases) that drives mouse Insc transcription in mES cells within a region >5 kb upstream of the mouse Insc transcription start site. We found that the transcription factor reticuloendotheliosis oncogene (c-Rel) bound to the minimum element and promoted mouse Insc expression in mES cells. In addition, short interfering RNA-mediated knockdown of either mouse INSC or c-Rel protein decreased mesodermal cell populations without affecting differentiation into the mesendoderm or endoderm. Furthermore, overexpression of mouse INSC rescued the mesoderm-reduced phenotype induced by knockdown of c-Rel. We propose that regulation of mouse Insc expression by c-Rel modulates cell fate decisions during mES cell differentiation.

The Metastasis Suppressor, N-MYC Downstream-regulated Gene-1 (NDRG1), Down-regulates the ErbB Family of Receptors to Inhibit Downstream Oncogenic Signaling Pathways.

N-MYC downstream-regulated gene-1 (NDRG1) is a potent growth and metastasis suppressor that acts through its inhibitory effects on a wide variety of cellular signaling pathways, including the TGF-ß pathway, protein kinase B (AKT)/PI3K pathway, RAS, etc. To investigate the hypothesis that its multiple effects could be regulated by a common upstream effector, the role of NDRG1 on the epidermal growth factor receptor (EGFR) and other members of the ErbB family, namely human epidermal growth factor receptor 2 (HER2) and human epidermal growth factor receptor 3 (HER3), was examined. We demonstrate that NDRG1 markedly decreased the expression and activation of EGFR, HER2, and HER3 in response to the epidermal growth factor (EGF) ligand, while also inhibiting formation of the EGFR/HER2 and HER2/HER3 heterodimers. In addition, NDRG1 also decreased activation of the downstream MAPKK in response to EGF. Moreover, novel anti-tumor agents of the di-2-pyridylketone class of thiosemicarbazones, namely di-2-pyridylketone 4,4-dimethyl-3-thiosemicarbazone and di-2-pyridylketone 4-cyclohexyl-4-methyl-3-thiosemicarbazone, which markedly up-regulate NDRG1, were found to inhibit EGFR, HER2, and HER3 expression and phosphorylation in cancer cells. However, the mechanism involved appeared dependent on NDRG1 for di-2-pyridylketone 4,4-dimethyl-3-thiosemicarbazone, but was independent of this metastasis suppressor for di-2-pyridylketone 4-cyclohexyl-4-methyl-3-thiosemicarbazone. This observation demonstrates that small structural changes in thiosemicarbazones result in marked alterations in molecular targeting. Collectively, these results reveal a mechanism for the extensive downstream effects on cellular signaling attributed to NDRG1. Furthermore, this study identifies a novel approach for the treatment of tumors resistant to traditional EGFR inhibitors.

Tubulin-binding agents down-regulate matrix metalloproteinase-2 and -9 in human hormone-refractory prostate cancer cells ­ a critical role of Cdk1 in mitotic entry.

Tubulin is an important target for anticancer therapy. Taxanes and vinca alkaloids are two groups of tubulin-binding agents in cancer chemotherapy. Besides tubulin binding, these groups of agents can also down-regulate protein levels of matrix metalloproteinase (MMP)-2 and -9, two important cancer-associated zinc-dependent endopeptidases in invasion and metastasis. However, the mechanism of action waits to be explored. In this study, protein levels but not mRNA expressions of MMP-2 and -9 were down-regulated by paclitaxel (a microtubule-stabilization agent), vincristine and evodiamine (two tubulin-depolymerization agents). These agents induced an increase of protein expression of cyclin B1, MPM2 (mitosis-specific phosphoprotein) and polo-like kinase (PLK) 1 phosphorylation. The data showed a negative relationship between the levels of mitotic proteins and MMP-2 and -9 expressions. MG132 (a specific cell-permeable proteasome inhibitor) blocked mitotic entry and arrested cell cycle at G2 phase, preventing down-regulation of MMP-2 and -9. Cell cycle synchronization experiments by thymidine block or nocodazole treatment showed that mitotic exit inhibited the down-regulation of MMP-2 and -9, confirming negative relationship between cell mitosis and protein levels of MMP-2 and -9 expressions. Cyclin-dependent kinase (Cdk) 1 is a key kinase in mitotic entry. Knockdown of Cdk1 almost completely inhibited the down-regulation of MMP-2 and -9 induced by tubulin-binding agents. In conclusion, the data suggest that mitotic entry and Cdk1 plays a central role in down-regulation of MMP-2 and -9 protein expressions. Tubulin-binding agents cause mitotic arrest and Cdk1 activation, which may contribute largely to the down-regulation of both MMP-2 and -9 expressions.

Cardiotrophin-1 stimulates lipolysis through the regulation of main adipose tissue lipases.

Cardiotrophin-1 (CT-1) is a cytokine with antiobesity properties and with a role in lipid metabolism regulation and adipose tissue function. The aim of this study was to analyze the molecular mechanisms involved in the lipolytic actions of CT-1 in adipocytes. Recombinant CT-1 (rCT-1) effects on the main proteins and signaling pathways involved in the regulation of lipolysis were evaluated in 3T3-L1 adipocytes and in mice. rCT-1 treatment stimulated basal glycerol release in a concentration- and time-dependent manner in 3T3-L1 adipocytes. rCT-1 (20 ng/ml for 24 h) raised cAMP levels, and in parallel increased protein kinase (PK)A-mediated phosphorylation of perilipin and hormone sensitive lipase (HSL) at Ser660. siRNA knock-down of HSL or PKA, as well as pretreatment with the PKA inhibitor H89, blunted the CT-1-induced lipolysis, suggesting that the lipolytic action of CT-1 in adipocytes is mainly mediated by activation of HSL through the PKA pathway. In ob/ob mice, acute rCT-1 treatment also promoted PKA-mediated phosphorylation of perilipin and HSL at Ser660 and Ser563, and increased adipose triglyceride lipase (desnutrin) content in adipose tissue. These results showed that the ability of CT-1 to regulate the activity of the main lipases underlies the lipolytic action of this cytokine in vitro and in vivo, and could contribute to CT-1 antiobesity effects.

Reversal of bortezomib resistance in myelodysplastic syndrome cells by MAPK inhibitors.

The myelodysplastic syndromes (MDS) comprise a heterogeneous group of malignant neoplasms with distinctive clinicopathological features. Currently, there is no specific approach for the treatment of MDS. Here, we report that bortezomib (BTZ), a proteasome inhibitor that has been used to treat plasma cell myeloma, induced G2/M phase cycle arrest in the MDS cell line SKM-1 through upregulation of Wee1, a negative regulator of G2/M phase transition. Treatment by BTZ led to reduced SKM-1 cell viability as well as increased apoptosis and autophagy. The BTZ-induced cell death was associated with reduced expression of p-ERK. To elucidate the implications of downregulation of p-ERK, we established the BTZ resistant cell line SKM-1R. Our data show that resistance to BTZ-induced apoptosis could be reversed by the MEK inhibitors U0126 or PD98059. Our results suggest that MAPK pathway may play an important role in mediating BTZ resistance.

Proteomic-based identification of multiple pathways underlying n-butylidenephthalide-induced apoptosis in LNCaP human prostate cancer cells.

Although numerous studies have shown the cancer-preventive properties of butylidenephthalide (BP), there is little report of BP affecting human prostate cancer cells. In the present study, proteomic-based approaches were used to elucidate the anticancer mechanism of BP in LNCaP human prostate cancer cells. BP treatment decreased the viability of LNCaP human prostate cancer cells in a concentration- and time-dependent manner, which was correlated with G0/G1 phase cell cycle arrest. Increased cell cycle arrest was associated with a decrease in the level of CCND1, CDK2, and PCNA proteins and an increase in the level of CDKN2A, CDKN1A, and SFN proteins. Proteomic studies revealed that among 48 differentially expressed proteins, 25 proteins were down-regulated and 23 proteins were up-regulated and these proteins fall into one large protein protein interaction network. Among these proteins, FAS, AIFM1, BIK, CYCS, SFN, PPP2R1A, CALR, HSPA5, DDIT3, and ERN1 are apoptosis and endoplasmic reticulum (ER) stress associated proteins. Proteomic data suggested that multiple signaling pathways including FAS-dependent pathway, mitochondrial pathway, and ER stress pathway are involved in the apoptosis induced by BP.

Hepatitis B virus X antigen and aflatoxin B1 synergistically cause hepatitis, steatosis and liver hyperplasia in transgenic zebrafish.

Aflatoxin B1 (AFB1) and the hepatitis B virus X antigen (HBx) are linked to the formation of liver diseases and hepatocellular carcinoma (HCC). The aim of this study was to investigate the synergistic effects between HBx and AFB1 in causing liver disorders using a transgenic zebrafish animal model. Histopathology, Periodic acid-Schiff (PAS) staining, Sirius red staining, TdT-mediated dUTP Nick End Labeling (TUNEL) assay, immunohistochemistry, and quantitative reverse transcriptase-polymerase chain reaction (Q-RT-PCR) were used to examine the livers of the HBx transgenic fish injected with AFB1. We found that HBx and AFB1 synergistically promoted steatosis as indicated by histopathological examinations and the increased expression of lipogenic factors, enzymes, and genes related to lipid metabolism. Moreover, treatment of AFB1 in HBx transgenic fish accelerated the development of liver hyperplasia and enhanced the expression of cell cycle related genes. PCNA was co-localized with active caspase 3 protein expression in HBx zebrafish liver samples and human HBV positive HCC samples by double fluorescence immunostaining. Finally, we found that in human patients with liver disease, significant glycogen accumulated in the inflammation, cirrhosis stage, and all cases of hepatocellular and cholangiocellular carcinoma showed a moderate cytoplasmic accumulation of glycogen. Our data demonstrated a synergistic effect of AFB1 and HBx on the regulation of lipid metabolism related genes and cell cycle/division-related genes which might contribute to enhanced steatosis and hyperplasia at 5.75months.

Mono-(2-ethylhexyl) phthalate induces oxidative stress and inhibits growth of mouse ovarian antral follicles.

Mono-(2-ethylhexyl) phthalate (MEHP) is the active metabolite of the most commonly used plasticizer, di-(2-ethylhexyl) phthalate, and is considered to be a reproductive toxicant. However, little is known about the effects of MEHP on ovarian antral follicles. Thus, the present study tested the hypothesis that MEHP inhibits follicle growth via oxidative stress pathways. The data indicate that MEHP increases reactive oxygen species (ROS) levels and inhibits follicle growth in antral follicles, whereas N-acetylcysteine (NAC; an antioxidant) restores ROS levels to control levels and rescues follicles from MEHP-induced inhibition of follicle growth. To further analyze the mechanism by which MEHP induces oxidative stress and inhibits follicle growth, the expression and activities of various key antioxidant enzymes (copper/zinc superoxide dismutase [SOD1], glutathione peroxidase [GPX], and catalase [CAT]) and the expression of key cell-cycle regulators (Ccnd2, Ccne1, and Cdk4) and apoptotic regulators (Bcl-2 and Bax) were compared in control and MEHP-treated follicles. The data indicate that MEHP inhibits the expression and activities of SOD1 and GPX; does not inhibit Cat expression; inhibits the expression of Ccnd2, Ccne1, Cdk4, and Bcl-2; but increases the expression of Bax compared to controls. Furthermore, NAC blocks these toxic effects of MEHP. Collectively, these data suggest that MEHP induces oxidative stress by disrupting the activities of antioxidant enzymes. This may lead to decreased expression of cell-cycle regulators and antiapoptotic regulators and increased expression of proapoptotic factors, which then may lead to inhibition of follicle growth.

Valproic acid inhibits proliferation of EB virus-infected natural killer cells.

There is no recognized treatment for Epstein-Barr virus (EBV)-associated natural killer (NK) cell lymphoproliferative disorders (LPDs). To determine the possibility of histone deacetylase inhibitors as a therapeutic tool for such disorders, we investigated the anti-proliferative effects of valproic acid (VPA) on two EBV-infected NK cell lines (KAI3 and NKED). VPA inhibited the growth of both lines in a dose- and time-dependent manner by inducing histone hyperacetylation. G1 cell cycle arrest was induced at 24 hours and was associated with increased expression of p21(WAF1), p27(Kip1), and cyclin E and decreased expression of cyclin D2, CDK4, and c-myc. Sub-G1 fractions were not significantly changed at 24 hours, whereas cleaved caspase-3 and cleaved PARP were already detected, and the extrinsic apoptotic pathway, determined by cleaved caspase-8, was activated. Finally, sub-G1 accumulation was increased after 72 hours following stimulation. These findings indicate that VPA might be a therapeutic option for EBV-associated NK-cell LPDs.

Arsenic trioxide induces G2/M arrest in hepatocellular carcinoma cells by increasing the tumor suppressor PTEN expression.

Arsenic trioxide (As(2)O(3)), an effective agent against acute promyelocytic leukemia, has been reported to inhibit the viability of solid tumors cell lines recently. The detailed molecular mechanism underlying the As(2)O(3)-induced inactivation of the cdc2 and possible functional role of PTEN in the observed G2/M arrest has yet to be elucidated. Here, we assessed the role of PTEN in regulation of As(2)O(3)-mediated G2/M cell cycle arrest in Hepatocellular carcinoma cell lines (HepG2 and SMMC7721). After 24 h following treatment, As(2)O(3) induced a concentration-dependent accumulation of cells in the G2/M phase of the cell cycle. The sustained G2/M arrest by As(2)O(3) is associated with decreased cdc2 protein and increased phospho-cdc2(Tyr15). As(2)O(3) treatment increased Wee1 levels and decreased phospho-Wee1(642). Moreover, As(2)O(3) substantially decreased the Ser473 and Thr308 phosphorylation of Akt and upregulated PTEN expression. Downregulation of PTEN by siRNA in As(2)O(3) -treated cells increased phospho-Wee1(Ser642) while decreased phospho-cdc2(Tyr15), resulting in decreased the G2/M cell cycle arrest. Therefore, induction of G2/M cell cycle arrest by As(2)O(3) involved upregulation of PTEN.

Functional characterization and targeted correction of ATM mutations identified in Japanese patients with ataxia-telangiectasia.

A recent challenge for investigators studying the progressive neurological disease ataxia-telangiectasia (A-T) is to identify mutations whose effects might be alleviated by mutation-targeted therapies. We studied ATM mutations in eight families of Japanese A-T patients (JPAT) and were able to identify all 16 mutations. The probands were compound heterozygotes in seven families, and one (JPAT2) was homozygous for a frameshift mutation. All mutations--four frameshift, two nonsense, four large genomic deletions, and six affecting splicing--were novel except for c.748C>T found in family JPAT6 and c.2639-384A>G found in family JPAT11/12. Using an established lymphoblastoid cell line (LCL) of patient JPAT11, ATM protein was restored to levels approaching wild type by exposure to an antisense morpholino oligonucleotide designed to correct a pseudoexon splicing mutation. In addition, in an LCL from patient JPAT8/9, a heterozygous carrier of a nonsense mutation, ATM levels could also be partially restored by exposure to readthrough compounds (RTCs): an aminoglycoside, G418, and a novel small molecule identified in our laboratory, RTC13. Taken together, our results suggest that screening and functional characterization of the various sorts of mutations affecting the ATM gene can lead to better identification of A-T patients who are most likely to benefit from rapidly developing mutation-targeted therapeutic technologies.

Antiapoptotic effects of roscovitine on camptothecin-induced DNA damage in neuroblastoma cells.

In the present study dopaminergic neuroblastoma B65 cells were exposed to Camptothecin (CPT) (0.5-10 µM), either alone or in the presence of roscovitine (ROSC). The results show that CPT induces apoptosis through the activation of ataxia telangiectasia mutated (ATM)-induced cell-cycle alteration in neuroblastoma B65 cells. The apoptotic process is mediated through the activation of cystein proteases, namely calpain/caspases. However, whereas a pan-caspase inhibitor, zVADfmk, inhibited CPT-mediated apoptosis, a calpain inhibitor, calpeptin, did not prevent cell death. Interestingly, CPT also induces CDK5 activation and ROSC (25 µM) blocked CDK5, ATM activation and apoptosis (as measured by caspase-3 activation). By contrast, selective inhibition of ATM, by KU55933, and non-selective inhibition, by caffeine, did not prevent CPT-mediated apoptosis. Thus, we conclude that CDK5 is activated in response to DNA damage and that CDK5 inhibition prevents ATM and p53ser15 activation. However, pharmacological inhibition of ATM using KU55933 and caffeine suggests that ATM inhibition by ROSC is not the only mechanism that might explain the anti-apoptotic effects of this drug in this apoptosis model. Our findings have a potential clinical implication, suggesting that combinatory drugs in the treatment of cancer activation should be administered with caution.