Amidic modification of valproic acid reduces skeletal teratogenicity in mice

BACKGROUND: The antiepileptic drug valproic acid (VPA) is well known to cause neural tube and skeletal defects in both humans and animals. The amidic VPA analogues valpromide (VPD) and valnoctamide (VCD) have much lower teratogenicity than VPA inducing exencephaly in mice. The objective of this study was to investigate the teratogenic effects of VPA, VPD, and VCD on the skeleton of NMRI mice. METHODS: Pregnant NMRI mice were given a single subcutaneous injection of VPA (400 and 800 mg/kg), VPD (800 mg/kg), or VCD (800 mg/kg) on the morning of gestation day (GD) 8. Cesarean section was carried out on GD 18. Live fetuses were double‐stained for bone and cartilage and their skeletons were examined. RESULTS: Significant increases in fetal loss and exencephaly rate were observed with VPA at 800 mg/kg compared to the vehicle control. There were no significant differences between either VPD or VCD and the control groups for any parameter at cesarean section. A number of abnormalities were dose‐dependently induced at high incidences by VPA in both the cartilage and bone of vertebrae, ribs and sternum. In contrast, lower frequencies of abnormality were exhibited with VPD and VCD than VPA in all skeletons affected by VPA. CONCLUSIONS: These findings clearly indicate that VPD and VCD are distinctly less teratogenic than VPA in the induction of not only neural tube defects, but also skeletal abnormalities. A structure‐teratogenicity relationship of VPA on the skeleton is suspected. Birth Defects Res B 71:47–53, 2004. © 2004 Wiley‐Liss, Inc.     

Effects of Ethylene Glycol Monomethyl Ether and Its Metabolite, 2‐Methoxyacetic Acid,on Organogenesis Stage Mouse Limbs In Vitro

Exposure to ethylene glycol monomethyl ether (EGME), a glycol ether compound found in numerous industrial products, or to its active metabolite, 2‐methoxyacetic acid (2‐MAA), increases the incidence of developmental defects. Using an in vitro limb bud culture system, we tested the hypothesis that the effects of EGME on limb development are mediated by 2‐MAA‐induced alterations in acetylation programming. Murine gestation day 12 embryonic forelimbs were exposed to 3, 10, or 30 mM EGME or 2‐MAA in culture for 6 days to examine effects on limb morphology; limbs were cultured for 1 to 24 hr to monitor effects on the acetylation of histones (H3K9 and H4K12), a nonhistone protein, p53 (p53K379), and markers for cell cycle arrest (p21) and apoptosis (cleaved caspase‐3). EGME had little effect on limb morphology and no significant effects on the acetylation of histones or p53 or on biomarkers for cell cycle arrest or apoptosis. In contrast, 2‐MAA exposure resulted in a significant concentration‐dependent increase in limb abnormalities. 2‐MAA induced the hyperacetylation of histones H3K9Ac and H4K12Ac at all concentrations tested (3, 10, and 30 mM). Exposure to 10 or 30 mM 2‐MAA significantly increased acetylation of p53 at K379, p21 expression, and caspase‐3 cleavage. Thus, 2‐MAA, the proximate metabolite of EGME, disrupts limb development in vitro, modifies acetylation programming, and induces biomarkers of cell cycle arrest and apoptosis     

Spatiotemporal activation of caspase‐dependent and ‐independent pathways in staurosporine‐induced apoptosis of p53wt and p53mt human cervical carcinoma cells

Background information. Caspase‐dependent and ‐independent death mechanisms are involved in apoptosis in a variety of human carcinoma cells treated with antineoplastic compounds. Our laboratory has reported that p53 is a key contributor of mitochondrial apoptosis in cervical carcinoma cells after staurosporine exposure. However, higher mitochondrial membrane potential dissipation and greater DNA fragmentation were observed in p53^wt (wild‐type p53) HeLa cells compared with p53^mt (mutated p53) C‐33A cells. Here, we have studied events linked to the mitochondrial apoptotic pathway. Results. Staurosporine can induce death of HeLa cells via a cytochrome c/caspase‐9/caspase‐3 mitochondrial‐dependent apoptotic pathway and via a delayed caspase‐independent pathway. In contrast with p53^wt cells, p53^mt C‐33A cells exhibit firstly caspase‐8 activation leading to caspase‐3 activation and Bid cleavage followed by cytochrome c release. Attenuation of PARP‐1 [poly(ADP‐ribose) polymerase‐1] cleavage as well as oligonucleosomal DNA fragmentation in the presence of z‐VAD‐fmk points toward a major involvement of a caspase‐dependent pathway in staurosporine‐induced apoptosis in p53^wt HeLa cells, which is not the case in p53^mt C‐33A cells. Meanwhile, the use of 3‐aminobenzamide, a PARP‐1 inhibitor known to prevent AIF (apoptosis‐inducing factor) release, significantly decreases staurosporine‐induced death in these p53^mt carcinoma cells, suggesting a preferential implication of caspase‐independent apoptosis. On the other hand, we show that p53, whose activity is modulated by pifithrin‐α, isolated as a suppressor of p53‐mediated transactivation, or by PRIMA‐1 (p53 reactivation and induction of massive apoptosis), that reactivates mutant p53, causes cytochrome c release as well as mitochondrio—nuclear AIF translocation in staurosporine‐induced apoptosis of cervical carcinoma cells. Conclusions. The present paper highlights that staurosporine engages the intrinsic mitochondrial apoptotic pathway via caspase‐8 or caspase‐9 signalling cascades and via caspase‐independent cell death, as well as through p53 activity.     

Characterization of valproic acid‐initiated homologous recombination

BACKGROUND : Valproic acid (VPA) is a frequently used antiepileptic agent and known teratogen. Previous research suggests that inhibition of histone deacetylases (HDACs) may play a role in VPA‐induced teratogenicity. We have also shown that VPA exposure leads to both an increase in reactive oxygen species (ROS) production and increased frequency of homologous recombination (HR). METHODS : In the present study, we evaluated the role of HDAC inhibition in VPA‐initiated HR to determine if HDAC inhibition could alter repair activity and/or cause DNA double‐strand breaks (DSBs), which would then initiate repair. Histone acetylation status was assessed to determine if VPA exposure led to HDAC inhibition in CHO 33 cells. RESULTS : Our results demonstrate that VPA (5 mM) exposure leads to increased acetylated histone H3 and H4 protein levels after 10 to 24 hr. Secondly, in our recombination assay where an artificial DNA DSB was induced in CHO 33 cells to assess repair activity, VPA exposure did not affect the repair activity of VPA‐initiated HR. Subsequently, to determine if VPA could increase susceptibility to DNA DSBs, the number of γ‐H2AX foci was assessed using immunocytochemistry and results revealed an increase in γ‐H2AX foci after 10‐ to 24‐hr exposure to VPA. CONCLUSIONS : Although we demonstrated the protective effect of polyethylene glycol‐catalase against VPA‐induced HR and the generation of intracellular ROS within 24 hr, we did not observed an increase in DNA oxidation. These studies suggest that HDAC inhibition and ROS signaling may play roles in DNA maintenance and cell‐cycle arrest in initiating DNA damage and repair. Birth Defects Res (Part B) 89:124–132, 2010. © 2010 Wiley‐Liss, Inc.     

Histone deacetylase inhibitors with a primary amide zinc binding group display antitumor activity in xenograft model

Histone deacetylase (HDAC) inhibition causes hyperacetylation of histones leading to differentiation, growth arrest and apoptosis of malignant cells, representing a new strategy in cancer therapy. Many of the known HDAC inhibitors (HDACi) that are in clinical trials possess a hydroxamic acid, that is a strong Zn²⁺ binding group, thereby inhibiting some of the class I and class II isoforms. Herein we describe the identification of a selective class I HDAC inhibitor bearing a primary carboxamide moiety as zinc binding group. This HDACi displays good antiproliferative activity against multiple cancer cell lines, and demonstrates efficacy in a xenograft model comparable to vorinostat.     

Sodium valproate potentiates staurosporine‐induced apoptosis in neuroblastoma cells via Akt/survivin independently of HDAC inhibition

Sodium valproate (VPA) has been recently identified as a selective class I histone deacetylase (HDAC) inhibitor and explored for its potential as an anti‐cancer agent. The anti‐cancer properties of VPA are generally attributed to its HDAC inhibitory activity indicating a clear overlap of these two actions, but the underlying mechanisms of its anti‐tumor effects are not clearly elucidated. The present study aimed to delineate the molecular mechanism of VPA in potentiating cytotoxic effects of anti‐cancer drugs with focus on inhibition of HDAC activity. Using human neuroblastoma cell lines, SK‐N‐MC, SH‐SY5Y, and SK‐N‐SH, we show that non‐toxic dose (2 mM) of VPA enhanced staurosporine (STS)‐induced cell death as assessed by MTT assay, PARP cleavage, hypodiploidy, and caspase 3 activity. Mechanistically, the effect of VPA was mediated by down regulation of survivin, an anti‐apoptotic protein crucial in resistance to STS‐mediated cytotoxicity, through Akt pathway. Knock down of class I HDAC isoforms remarkably inhibited HDAC activity comparable with that of VPA but had no effect on STS‐induced apoptosis. Moreover, MS‐275, a structurally distinct class I HDAC inhibitor did not affect STS‐mediated apoptosis, nor decrease the levels of survivin and Akt. Valpromide (VPM), an amide analog of VPA that does not inhibit HDAC also potentiated cell death in NB cells associated with decreased survivin and Akt levels suggesting that HDAC inhibition might not be crucial for STS‐induced apoptosis. The study provides new information on the possible molecular mechanism of VPA in apoptosis that can be explored in combination therapy in cancer. J. Cell. Biochem. 114: 854–863, 2013. © 2012 Wiley Periodicals, Inc.     

A proapoptotic effect of valproic acid on progenitors of embryonic stem cell-derived glutamatergic neurons

Valproic acid (VPA) is a branched-chain saturated fatty acid with a long history of clinical use as an antiepileptic drug (AED). VPA is also known to inhibit histone deacetylases (HDACs) and to cause diverse effects on neural progenitor cells (NPCs) and neurons. Although the neuroprotective or neurodestructive effects of VPA have been investigated in heterogeneous cell populations, in this study, we used homogeneous populations of NPCs and glutamatergic cortical pyramidal neurons, which were differentiated from embryonic stem (ES) cells. At therapeutic concentrations, VPA had a proapoptotic effect on ES cell-derived NPCs of glutamatergic neurons, but not on their progeny. This effect of VPA most likely occurred through the inhibition of HDACs, because similar phenotypes were observed following treatment with other HDAC inhibitors (HDACis) such as trichostatin A and sodium butyrate. The proapoptotic phenotype was not observed when cells were exposed to a structural analog of VPA, valpromide (VPM), which has the same antiepileptic effect as VPA, but does not inhibit HDACs. Western blotting confirmed that treatment with HDACis, but not VPM, significantly increased the levels of histone H3 acetylation in NPCs. HDACi treatments did not affect the survival of neurons, although the acetylation levels were increased to a limited extent. These results, which are based on a homogeneous culture system, suggest that VPA inhibits HDAC activity and induces the apoptosis of NPCs that are fated to differentiate into glutamatergic neurons. The dose-dependent effects of VPA both on apoptosis and hyperacetylation of histone H3 in NPCs supported this notion. These cell type- and differentiation stage-specific effects of VPA imply that dysfunction of HDACs during pregnancy significantly increase the risk of congenital malformations associated with VPA administration.     

p53 signalling controls cell cycle arrest and caspase‐independent apoptosis in macrophages infected with pathogenic Leptospira species

Pathogenic Leptospira species, the causative agents of leptospirosis, have been shown to induce macrophage apoptosis through caspase‐independent, mitochondrion‐related apoptosis inducing factor (AIF) and endonuclease G (EndoG), but the signalling pathway leading to AIF/EndoG‐based macrophage apoptosis remains unknown. Here we show that infection of Leptospira interrogans caused a rapid increase in reactive oxygen species (ROS), DNA damage, and intranuclear foci of 53BP1 and phosphorylation of H2AX (two DNAdamage indicators) in wild‐type p53‐containing mouse macrophages and p53‐deficient human macrophages. Most leptospire‐infected cells stayed at the G₁ phase, whereas depletion or inhibition of p53 caused a decrease of the G₁‐phase cells and the early apoptotic ratios. Infection with spirochaetes stimulated a persistent activation of p53 and an early activation of Akt through phosphorylation. The intranuclear translocation of p53, increased expression of p53‐dependent p21^Cip1/WAF1 and pro‐apoptotic Bcl‐2 family proteins (Bax, Noxa and Puma), release of AIF and EndoG from mitochondria, and membrane translocation of Fas occurred during leptospire‐induced macrophage apoptosis. Thus, our study demonstrated that ROS production and DNA damage‐dependent p53‐Bax/Noxa/Puma‐AIF/EndoG signalling mediates the leptospire‐induced cell cycle arrest and caspase‐independent apoptosis of macrophages.     

Edwardsiella piscicida type III protein EseJ suppresses apoptosis through down regulating type 1 fimbriae,which stimulate the cleavage of caspase‐8

The type III secretion system effector EseJ plays a regulatory role inside bacteria. It suppresses the adherence of Edwardsiella piscicida (E. piscicida) to host epithelial cells by down regulating type 1 fimbriae. In this study, we observed that more macrophages infected with ΔeseJ strain of E. piscicida detached as compared with those infected with the wild‐type (WT) strain. Terminal deoxynucleotidyl transferase dUTP nick‐end labelling (TUNEL) staining and cleaved caspase‐3 examination revealed that the detachment is due to increased apoptosis, suggesting that EseJ suppresses macrophage apoptosis. However, apoptosis inhibition by EseJ is not relative to a type III secretion system (T3SS) and is not related to EseJ's translocation. Since EseJ negatively regulates type 1 fimbriae, murine J774A.1 cells were infected with ΔeseJΔfimA or ΔeseJΔfimH strains. It was demonstrated that ΔeseJ stimulates macrophage apoptosis through type 1 fimbriae. Moreover, we found that infecting J774A.1 cells with the ΔeseJ strain increased levels of cleaved caspase‐8, caspase‐9, and caspase‐3, demonstrating that EseJ inhibits apoptosis through either an extrinsic or a combination of extrinsic and intrinsic pathways. Pre‐treatment of macrophages with caspase‐8 inhibitor prior to infection with the ΔeseJ strain decreased the levels of cleaved caspase‐8, caspase‐9, and caspase‐3, indicating that the ΔeseJ strain stimulates apoptosis, mainly through an extrinsic pathway by up regulating type 1 fimbriae. Zebrafish larvae or blue gourami fish infected with the ΔeseJ strain consistently exhibited higher apoptosis than those infected with the E. piscicida WT strain or ΔeseJΔfimA strain. Taken together, we revealed that the T3SS protein EseJ of E. piscicida inhibits host apoptosis, mainly through an extrinsic pathway by down regulating type 1 fimbriae.     

Association of TP53 mutation, p53 overexpression, and p53 codon 72 polymorphism with susceptibility to apoptosis in adult patients with diffuse astrocytomas

Clarification of TP53 alterations is important to understand the mechanisms underlying the development of diffuse astrocytomas. It has been suggested that the alleles of TP53 at codon 72 differ in their ability to induce apoptosis in human cancers. The aim of this study was to analyze the possible association of TP53 mutation, p53 overexpression, and p53 codon 72 polymorphism with susceptibility to apoptosis in adult Brazilian patients with diffuse astrocytomas. We analyzed 56 surgical specimens of diffuse astrocytomas for alterations of TP53, using polymerase chain reaction single-strand conformation polymorphism (PCR-SSCP) direct sequencing. p53 and cleaved caspase 3 protein expression were assessed by immunohistochemistry. We found TP53 mutations in 19.6% (11 out of 56) of tumors tested, with the lowest mutation rate found in the cases of glioblastomas (8.8%) (p = 0.03). Only 16.1% of tumors tested showed cleaved caspase 3-positive staining, demonstrating that apoptosis is very inhibited in these tumors. All tumors having TP53 mutation and p53 accumulation had no expression of cleaved caspase 3. Additionally, no association was observed in tumors having proline and arginine alleles and expression of cleaved caspase 3. We concluded that clarification of the TP53 alterations allows a better understanding of the mechanisms involved in the progression of diffuse astrocytomas, and the allele status at codon 72 was not associated with apoptosis in these tumors.     

Valproic acid stimulates proliferation of glial precursors during cortical gliogenesis in developing rat

Valproic acid (VPA) is a neurotherapeutic drug prescribed for seizures, bipolar disorder, and migraine, including women of reproductive age. VPA is a well‐known teratogen that produces congenital malformations in many organs including the nervous system, as well as later neurodevelopmental disorders, including mental retardation and autism. In developing brain, few studies have examined VPA effects on glial cells, particularly astrocytes. To investigate effects on primary glial precursors, we developed new cell culture and in vivo models using frontal cerebral cortex of postnatal day (P2) rat. In vitro, VPA exposure elicited dose‐dependent, biphasic effects on DNA synthesis and proliferation. In vivo VPA (300 mg/kg) exposure from P2 to P4 increased both DNA synthesis and cell proliferation, affecting primarily astrocyte precursors, as >75% of mitotic cells expressed brain lipid‐binding protein. Significantly, the consequence of early VPA exposure was increased astrocytes, as both S100‐β+ cells and glial fibrillary acidic protein were increased in adolescent brain. Molecularly, VPA served as an HDAC inhibitor in vitro and in vivo as enhanced proliferation was accompanied by increased histone acetylation, whereas it elicited changes in culture in cell‐cycle regulators, including cyclin D1 and E, and cyclin‐dependent kinase (CDK) inhibitors, p21 and p27. Collectively, these data suggest clinically relevant VPA exposures stimulate glial precursor proliferation, though at higher doses can elicit inhibition through differential regulation of CDK inhibitors. Because changes in glial cell functions are proposed as mechanisms contributing to neuropsychiatric disorders, these observations suggest that VPA teratogenic actions may be mediated through changes in astrocyte generation during development. © 2015 Wiley Periodicals, Inc. Develop Neurobiol 76: 780–798, 2016     

DNA‐PK suppresses a p53‐independent apoptotic response to DNA damage

p53 is required for DNA damage‐induced apoptosis, which is central to its function as a tumour suppressor. Here, we show that the apoptotic defect of p53‐deficient cells is nearly completely rescued by inactivation of any of the three subunits of the DNA repair holoenzyme DNA‐dependent protein kinase (DNA‐PK). Intestinal crypt cells from p53 nullizygous mice were resistant to radiation‐induced apoptosis, whereas apoptosis in DNA‐PK_cs/p53, Ku80/p53 and Ku70/p53 double‐null mice was quantitatively equivalent to that seen in wild‐type mice. This p53‐independent apoptotic response was specific to the loss of DNA‐PK, as it was not seen in ligase IV (Lig4)/p53 or ataxia telangiectasia mutated (Atm)/p53 double‐null mice. Furthermore, it was associated with an increase in phospho‐checkpoint kinase 2 (CHK2), and cleaved caspases 3 and 9, the latter indicating engagement of the intrinsic apoptotic pathway. This shows that there are two separate, but equally effective, apoptotic responses to DNA damage: one is p53 dependent and the other, engaged in the absence of DNA‐PK, does not require p53.     

Involvement of RPL11 in the enhancement of P53 stability by a podophyllum derivative,a topoisomerase II inhibitor

In previous work, we presented experimental and theoretical evidence that D‐3F or 4‐N‐(2‐Amino‐3‐fluoropyridine)‐4‐deoxidation‐4′‐demethylepipofophyllotoxin induced G₂/M phase arrest and apoptosis, purportedly by increasing the expression of P53. However, the precise mechanism of D‐3F action is currently unknown. Here, we investigated the mechanism by which D‐3F treatment induces increased expression of P53. This study showed that D‐3F definitively inhibited the activity of topoisomerase II in a dose‐dependent manner and resulted in DNA damage. The results were in overall agreement with modeling and docking studies performed on D‐3F . In addition, D‐3F increased the levels of P53 and P21 in HeLa cells in a dose‐dependent manner, this in turn prolonged the half‐life of P53. Taken together, these data suggested that D‐3F ‐mediated transient enhancement of P53 stabilization may be critical for the P53/P21 signalling pathway leading to G₂/M phase arrest on HeLa cells. Furthermore, D‐3F downregulated the phosphorylation of E3 ubiquitin‐protein ligase murine double minute 2 (Mdm2) at Ser166, inhibited Mdm2‐mediated ubiquitination of P53, and released 60S ribosomal protein L11 (RPL11) from the nucleolus into the nucleoplasm. To conclude, the topoisomerase II inhibitor D‐3F causes P53 to accumulate in HeLa cell lines by enhancing its stability as a result of DNA‐damage induced RPL11 relocalization and subsequent blocking of the P53‐Mdm2 feedback loop.     

Enantioselective synthesis and teratogenicity of propylisopropyl acetamide,a CNS‐active chiral amide analogue of valproic acid

Propylisopropyl acetamide (PID), an amide analogue of the major antiepileptic drug valproic acid (VPA), possesses favorable anticonvulsant and CNS properties. PID contains one chiral carbon atom and therefore exists in two enantiomeric forms. The purpose of this work was to synthesize the two PID enantiomers and evaluate their enantiospecific teratogenicity. Enantioselective synthesis of PID enantiomers was achieved by coupling valeroyl chloride with optically pure (4S)‐ and (4R)‐benzyl‐2‐oxazolidinone chiral auxiliaries. The two oxazolidinone enolates were alkylated with isopropyl triflate, hydrolyzed, and amidated to yield (2R)‐ and (2S)‐PID. These two PID enantiomers were obtained with excellent enantiomeric purity, exceeding 99.4%. Unlike VPA, both (2R)‐ and (2S)‐PID failed to exert teratogenic effects in NMRI mice following a single 3 mmol/kg subcutaneous injection. From this study we can conclude that individual PID enantiomers do not demonstrate stereoselective teratogenicity in NMRI mice. Due to its better anticonvulsant activity than VPA and lack of teratogenicity, PID (in a stereospecific or racemic form) has the potential to become a new antiepileptic and CNS drug. Chirality 11:645–650, 1999. © 1999 Wiley‐Liss, Inc.     

Inhibition of histone deacetylase activity on specific embryonic tissues as a new mechanism for teratogenicity

BACKGROUND: the inhibition of histone deacetylase (HDAC) has been reported as an effective mechanism on therapy in neoplastic diseases. Among HDAC inhibitors, Trichostatin A (TSA) and Valproic Acid (VPA) prevent the tumorigenesis in rodent and human models. Malformations as neural tube and axial skeletal defects are well-known VPA side effects. Recent hypotheses suggest the HDAC inhibitor activity as the teratogenic mechanism of VPA. The teratogenic potency of TSA is, at the moment, unknown. The aim of the present work is to investigate the HDAC inhibition on embryos exposed in utero to TSA or VPA and to compare the teratogenic potential of these two molecules on the axial skeleton morphogenesis. METHODS: Pregnant CD mice were i.p. treated on day 8 post coitum (9.00 a.m.) with 400 mg/kg VPA or with 0, 2, 4, 8, 16 mg/kg TSA. Embryos explanted 1 hr after the treatment from some females exposed to 400 mg/kg VPA or to 16 mg/kg TSA were processed for Western blotting and immunohistochemical analysis, in order to evaluate the histone hyperacetylation in the total embryo homogenates and to visualize the hyperacetylated tissues. Foetuses at term were processed for skeletal examination. RESULTS: Both VPA and TSA were able to induce hyperacetylation on embryos, specifically at the level of the caudal neural tube and of somites. At term, TSA showed teratogenic effects at the axial skeleton, quite similar to those observed after VPA exposure. CONCLUSIONS: In conclusion, both VPA and TSA are teratogenic in mice. A direct correlation between somite hyperacetylation and axial abnormalities could suggest the HDAC inhibition as the mechanism of the teratogenic effects.