Apoptosis In Primary Cultures Research Articles

A novel anti-apoptotic role for Cdc42/ACK-1 signaling in neurons

Neurodegenerative diseases such as amyotrophic lateral sclerosis, Alzheimer's and Parkinson's disease are caused by a progressive and aberrant destruction of neurons in the brain and spinal cord. These disorders lack effective long-term treatments that impact the underlying mechanisms of pathogenesis and as a result, existing options focus primarily on alleviating symptomology. Dysregulated programmed cell death (i.e., apoptosis) is a significant contributor to neurodegeneration, and is controlled by a number of different factors. Rho family GTPases are molecular switches with recognized importance in proper neuronal development and migration that have more recently emerged as central regulators of apoptosis and neuronal survival. Here, we investigated a role for the Rho GTPase family member, Cdc42, and its downstream effectors, in neuronal survival and apoptosis. We initially induced apoptosis in primary cultures of rat cerebellar granule neurons (CGNs) by removing both growth factor-containing serum and depolarizing potassium from the cell medium. We then utilized both chemical inhibitors and adenoviral shRNA targeted to Cdc42 to block the function of Cdc42 or its downstream effectors under either control or apoptotic conditions. Our in vitro studies demonstrate that functional inhibition of Cdc42 or its downstream effector, activated Cdc42-associated tyrosine kinase-1 (ACK-1), had no adverse effects on CGN survival under control conditions, but significantly sensitized neurons to cell death under apoptotic conditions. In conclusion, our results suggest a key pro-survival role for Cdc42/ACK-1 signaling in neurons, particularly in regulating neuronal susceptibility to pro-apoptotic stress such as that observed in neurodegenerative disorders.

PCB153 reduces apoptosis in primary cultures of murine pituitary cells through the activation of NF-κB mediated by PI3K/Akt

Polychlorinated biphenyls (PCBs) are persistent pollutants involved in human tumorigenesis. PCB153 is a ubiquitous non-dioxin-like PCB with proliferative and anti-apoptotic effects. To explore the impact of PCB153 in the survival of pituitary cells, we exposed murine pituitary primary cells to PCB153 10 μM for 24 h. Apoptosis was assessed by RT-qPCR, Western-blot, immunoprecipitation, caspase activity, and immunofluorescence. We found that PCB153 decreased pituitary apoptosis through both the extrinsic and intrinsic pathways. PCB153 reduced the level of the pro-apoptotic protein p38-MAPK. Otherwise, PCB153 activated PI3K/Akt and Erk1/2 pathways and enhanced the expression and nuclear translocation of NF-κB. Cotreatments with specific inhibitors revealed that only PI3K/Akt changed the caspase−3 expression and NF-κB activation induced by PCB153. Also, PCB153 decreased the expression of the pro-apoptotic and pro-senescent cyclins p53 and p21. In summary, exposure to PCB153 leads to a downregulation of apoptosis in the pituitary driven by a PI3K/Akt-mediated activation of NF-κB.

New selective thiolate gold(i) complexes inhibit the proliferation of different human cancer cells and induce apoptosis in primary cultures of mouse colon tumors.

New thiolate gold(i) complexes with P(NMe2)3 (HMPT) as a phosphane group [Au(SR)(HMPT)] (SR = Spy, Spyrim, SMe2pyrim, Sbenzothiazole, Sthiazoline, Sbenzimidazole and 2-thiouracil) have been synthesized. All of them have been characterized, including X-ray studies of complexes with SMe2pyrim, Sbenzothiazole and 2-thiouracil moieties. In addition, their potential application as anticancer drugs has been analyzed by determining their pharmacokinetic activities (water solubility, cell permeability and BSA transport protein affinity). Based on the good results of these experiments, we carried out the studies of cell viability with our compounds on different cell lines (A2780, A2780R and Caco-2/TC7 cells), showing higher cytotoxic activity than cisplatin in all cases. Besides, two of the synthesized complexes with Sbenzimidazole and 2-thiouracil groups exhibit specific selectivity for cancerous Caco-2 cells and are considered as potential candidates for anticancer drugs. These complexes were able to induce a strong inhibition of the thioredoxin reductase (TrxR) protein and oxidative damage in membrane lipids. Additional studies in primary cultures of mouse colon tumors showed that these two complexes are proapoptotic upon exposure to phosphatidylserine. Based on our results, we conclude that two of our thiolate gold(i) complexes are good and effective candidates for use in chemotherapy.

Phenolic Compounds Isolated from Calea uniflora Less. Promote Anti-Inflammatory and Antioxidant Effects in Mice Neutrophils (Ex Vivo) and in Mice Pleurisy Model (In Vivo).

The literature shows that phenolic compounds possess important antioxidant and anti-inflammatory activities; however, the mechanism underlying these effects is not elucidated yet. The genus Calea is used in folk medicine to treat rheumatism, respiratory diseases, and digestive problems. In this context, some phenolic compounds were isolated with high purity from Calea uniflora Less. and identified as noreugenin (NRG) and α-hydroxy-butein (AH-BU). The aim of this study was to analyze the effect of these compounds on cell viability, the activity of myeloperoxidase (MPO), and apoptosis of mouse neutrophils using ex vivo tests. Furthermore, the effect of these compounds on the cytokines, interleukin 1 beta (IL-1β), interleukin 17A (IL-17A), and interleukin 10 (IL-10), and oxidative stress was investigated by analyzing lipid peroxidation (the concentration of thiobarbituric acid reactive substances (TBARS)) and activities of antioxidant enzymes, superoxide dismutase (SOD), catalase (CAT), and glutathione S-transferase (GST), using a murine model of neutrophilic inflammation. The NRG and AH-BU reduce MPO activity and increase neutrophil apoptosis (p < 0.05). These compounds reduced the generation of oxygen reactive species and IL-1β and IL-17A levels but increased IL-10 levels (p < 0.05). This study demonstrated that NRG and AH-BU show a significant anti-inflammatory effect by inhibiting the MPO activity and increasing neutrophil apoptosis in primary cultures of mouse neutrophils. These effects were at least partially associated with blocking reactive species generation, inhibiting IL-1β and IL-17A, and increasing IL-10 levels.

CaMKII is involved in subcellular Ca2+ redistribution-induced endoplasmic reticulum stress leading to apoptosis in primary cultures of rat proximal tubular cells exposed to lead.

Lead (Pb) is a known nephrotoxic element. Recently we have proved that subcellular Ca2+ redistribution is involved in Pb-induced apoptosis in primary cultures of rat proximal tubular (rPT) cells, but the underlying mechanism remains to be elucidated. Firstly, data showed that Pb triggers endoplasmic reticulum (ER) stress response in rPT cells, as evidenced by the elevations of ER stress markers. Moreover, pharmacological modulation of Ca2+ mobilization in ER and cytoplasm with three chemicals (2-APB or TG or BAPTA-AM) can effectively increase or decrease the protein expression of ER stress markers in Pb-exposed rPT cells, demonstrating that Pb-induced ER stress is Ca2+-dependent. We found that Pb stimulates phosphorylation of calcium/calmodulin-dependent protein kinase II (CaMKII) to activate its activity. Meanwhile, inhibition of CaMKII with KN93 or KN62 attenuated Pb-activated caspase-12 and CCAAT/enhancer-binding protein homologous protein (CHOP) in rPT cells, demonstrating that CaMKII activation promoted ER stress in rPT cells. Likewise, Pb-induced apoptosis can be effectively inhibited by CaMKII inhibitor KN93 or KN62. Furthermore, co-treatment with KN93 or KN62 significantly reversed Pb-induced ER Ca2+ release and concomitant intracellular Ca2+ overload in rPT cells. In summary, these results expound the mechanisms involving in ER stress, Ca2+ dyshomeostasis and activated CaMKII, which all contribute to Pb-induced apoptosis. CaMKII acts as a critical mediator of ER stress and associated apoptosis via regulating intracellular Ca2+ mobilization from ER to cytoplasm.

A Role for Progesterone-Regulated sFRP4 Expression in Uterine Leiomyomas.

Context:Despite progesterone’s key role in uterine smooth muscle tumorigenesis, the mechanisms by which it promotes the growth of uterine leiomyomas remain poorly understood.Objective:The aim of this study was to identify gene products mediating the effects of progesterone in uterine leiomyomas.Design:Gene expression profiling was used to identify putative progesterone-regulated genes differentially expressed in uterine leiomyomas, which were then studied in vitro.Methods:Gene expression was comprehensively profiled with the Illumina WG BeadChip (version 2.6) and analyzed with a bioinformatic algorithm that integrates known protein–protein interactions. Genomic binding sites for progesterone receptor (PR) were interrogated by chromatin immunoprecipitation—quantitative polymerase chain reaction (ChIP-qPCR). Small interfering RNA was used to study gene function in primary cell lines.Results:Our analyses identified secreted Frizzled-related protein 4 (sFRP4) as a key gene product functionally linked to PR activation whose expression was 2.6 times higher in leiomyomas than myometrium (n = 26, P < 0.01) and 2.5 times higher during the proliferative phase of the menstrual cycle (n = 26, P < 0.01). Direct binding between PR and sFRP4 promoter was observed by ChIP-qPCR. Robust overexpression of sFRP4 was also observed in primary cultures derived from leiomyoma. Progesterone preferentially inhibited sFRP4 expression and secretion in leiomyoma cultures in a dose-dependent manner sensitized by estradiol. Knockdown of sFRP4 inhibited proliferation and apoptosis in primary cultures of both myometrium and leiomyoma.Conclusions:Overexpression of sFRP4 is a robust, progesterone-regulated feature of leiomyomas that increases smooth muscle proliferation. More work is needed to elucidate how progesterone’s ability to modulate sFRP4 expression contributes to uterine smooth muscle tumorigenesis.

Cathepsin L silencing increases As2O3 toxicity in malignantly transformed pilocytic astrocytoma MPA58 cells by activating caspases 3/7

Low-grade, pilocytic astrocytomas are treated by resection, but additional therapy is necessary for those tumors with anaplastic features. Arsenic trioxide (As2O3) is emerging as an effective chemotherapeutic agent for treatment of malignant glioblastoma multiforme, where Cathepsin L silencing enables lower, less harmful As2O3 concentrations to achieve the desired cytotoxic effect. Here, we evaluated the effects of As2O3 combined with stable Cathepsin L shRNA silencing on cell viability/metabolic activity, and apoptosis in primary cultures of recurrent malignantly transformed pilocytic astrocytoma (MPA). These cells expressed high Cathepsin L levels, and when grown as monolayers and spheroids, they were more resistant to As2O3 than the U87MG glioblastoma cell line. Caspases 3/7 activity in MPA58 spheroids was not significantly affected by As2O3, possibly due to higher chemoresistance of primary biopsy tissue of less malignant astrocytoma versus the malignant U87MG cell line. However, As2O3 treatment was cytotoxic to MPA spheroids after silencing of Cathepsin L expression. While Cathepsin L silencing only slightly decreased the live/dead cell ratio in As2O3-treated MPA-si spheroids under our experimental conditions, there was an increase in As2O3-mediated apoptosis in MPA-si spheroids, as indicated by elevated caspases 3/7 activity. Therefore, Cathepsin L silencing by gene manipulation can be applied when a more aggressive approach is needed in treatment of pilocytic astrocytomas with anaplastic features.

MTOR inhibitors response and mTOR pathway in pancreatic neuroendocrine tumors.

Medical therapy of pancreatic neuroendocrine tumors (P-NET) may take advantage of Everolimus treatment. However, the extent of therapeutic response cannot be predicted. This study was aimed to identify the possible predictive markers of response to Everolimus in P-NET. We found that Everolimus reduced the cell viability and induced apoptosis in primary cultures of 6 P-NET (P-NET-R), where the proliferative and antiapoptotic effects of IGF1 were blocked by Everolimus. On the contrary, 14 P-NET primary cultures (P-NET-NR) were resistant to Everolimus and IGF1, suggesting an involvement of PI3K/AKT/mTOR pathway in the mechanism of resistance. The response to Everolimus in vitro was associated with an active AKT/mTOR pathway and seemed to be associated with a greater clinical aggressiveness. In addition, a patient sensitive to Everolimus in vitro was sensitive to this drug in vivo also and showed a positive p-AKT immunohistochemistry (IHC) at tissue level. Similarly, a patient resistant to Everolimus treatment after surgery was not sensitive to the drug in vitro and had a negative p-AKT IHC staining. Therefore, present data confirm that P-NET primary cultures may be considered a model for testing medical treatment efficacy and that IHC characterization of p-AKT might help in identifying human P-NET who can benefit from Everolimus treatment. These data encourage conducting a prospective multicenter study involving different groups of P-NET patients treated with Everolimus.

Time-Dependent Changes in Apoptosis Upon Autophagy Inhibition in Astrocytes Exposed to Oxygen and Glucose Deprivation.

Recent studies have implicated the role of autophagy in brain ischemia pathophysiology. However, it remains unclear whether autophagy activation is protective or detrimental to astrocytes undergoing ischemic stress. This study evaluated the influence of ischemia-induced autophagy on cell death and the course of intrinsic and extrinsic apoptosis in primary cultures of rat cortical astrocytes exposed to combined oxygen-glucose deprivation (OGD). The role of autophagy was assessed by pharmacological inhibition with 3-methyladenine (3-MA). Cell viability was evaluated by measuring LDH release and through the use of the alamarBlue Assay. Apoptosis and necrosis were determined by fluorescence microscopy after Hoechst 33,342 and propidium iodide staining, respectively. The levels of apoptosis-related proteinswere analyzed by immunoblotting. The downregulation of autophagy during OGD resulted in decreased cell viability and time-dependent changes in levels of apoptosis and necrosis. After short-term OGD (1, 4h), cells treated with 3-MA showed higher level of cleaved caspase3 compared with control cells. This result was consistent with an evaluation of apoptotic cell number by fluorescence microscopy. However, after prolonged exposure to OGD (8, 24h), thenumber ofapoptotic astrocytes (microscopically evaluated)did not differ or was even lower (as marked by caspase 3) in the presence of the autophagy inhibitorin comparison tothe control. A higher level of necrosis was observed in 3-MA-treated cells compared to non-treated cells after 24h OGD. The downregulation of autophagy caused time-dependent changes in both extrinsic (cleaved caspase 8, TNFα) and intrinsic (cleaved caspase 9) apoptotic pathways. Our results strongly indicate that the activation of autophagy in astrocytes undergoing ischemic stress is an adaptive mechanism, which allows for longer cell survival by delaying the initiation of apoptosis and necrosis.

Differential Gene Expression Patterns in Chicken Cardiomyocytes during Hydrogen Peroxide-Induced Apoptosis.

Hydrogen peroxide (H2O2) is both an exogenous and endogenous cytotoxic agent that can reliably induce apoptosis in numerous cell types for studies on apoptosis signaling pathways. However, little is known of these apoptotic processes in myocardial cells of chicken, a species prone to progressive heart failure. Sequencing of mRNA transcripts (RNA-Seq) allows for the identification of differentially expressed genes under various physiological and pathological conditions to elucidate the molecular pathways involved, including cellular responses to exogenous and endogenous toxins. We used RNA-seq to examine genes differentially expressed during H2O2-induced apoptosis in primary cultures of embryonic chicken cardiomyocytes. Following control or H2O2 treatment, RNA was extracted and sequencing performed to identify novel transcripts up- or downregulated in the H2O2 treatment group and construct protein−protein interaction networks. Of the 19,268 known and 2,160 novel transcripts identified in both control and H2O2 treatment groups, 4,650 showed significant differential expression. Among them, 55.63% were upregulated and 44.37% downregulated. Initiation of apoptosis by H2O2 was associated with upregulation of caspase-8, caspase-9, and caspase-3, and downregulation of anti-apoptotic genes API5 and TRIA1. Many other differentially expressed genes were associated with metabolic pathways (including ‘Fatty acid metabolism’, ‘Alanine, aspartate, and glutamate metabolism’, and ‘Biosynthesis of unsaturated fatty acids’) and cell signaling pathways (including ‘PPAR signaling pathway’, ‘Adipocytokine signaling pathway’, ‘TGF-beta signaling pathway’, ‘MAPK signaling pathway’, and ‘p53 signaling pathway’). In chicken cardiomyocytes, H2O2 alters the expression of numerous genes linked to cell signaling and metabolism as well as genes directly associated with apoptosis. In particular, H2O2 also affects the biosynthesis and processing of proteins and unsaturated fatty acids. These results highlight the value of RNA-seq for revealing unexpected molecular contributors to oxidative stress responses, thereby identifying novel potential therapeutic targets.

Mitochondrial permeability transition and its regulatory components are implicated in apoptosis of primary cultures of rat proximal tubular cells exposed to lead.

Previous studies have already demonstrated that mitochondria play a key role in Pb-induced apoptosis in primary cultures of rat proximal tubular (rPT) cells. To further clarify the underlying mechanism of Pb-induced mitochondrial apoptosis, this study was designed to investigate the role of mitochondrial permeability transition (MPT) and its regulatory components in Pb-induced apoptosis in rPT cells. Mitochondrial permeability transition pore (MPTP) opening together with disruption of mitochondrial ultrastructure, translocation of cytochrome c from mitochondria to cytoplasm and subsequent caspase-3 activation were observed in this study, suggesting that MPT is involved in Pb-induced apoptosis in rPT cells. Simultaneously, Pb-induced caspase-3 activation and apoptosis can be significantly inhibited by three MPTP inhibitors (CsA, DIDS, BA), which target different regulatory components of MPTP (Cyp-D, VDAC, ANT), respectively, demonstrating that Cyp-D, VDAC and ANT participate in MPTP regulation during lead exposure. Moreover, decreased ATP levels and increased ADP/ATP ratio induced by lead treatment can be significantly reversed by BA, indicating that Pb-mediated ANT dysfunction resulted in ATP depletion. In addition, up-regulation of VDAC-1, ANT-1 together with down-regulation of Cyp-D, VDAC-2 and ANT-2 at both the levels of transcription and translation were revealed in rPT cells under lead exposure conditions. In conclusion, Pb-mediated mitochondrial apoptosis in rPT cells is dependent on MPTP opening. Different expression levels in each isoform of three regulatory components contribute to alteration in their functions, which may promote the MPTP opening.

Duck tembusu virus and its envelope protein induce programmed cell death.

The cytopathic effect produced in cells infected with duck tembusu virus (DTMUV) suggests that this emerging virus may induce apoptosis in primary cultures of duck embryo fibroblasts (DEF). Here, we present evidence that DTMUV infection of cultured cells activates apoptosis and that the ability of DTMUV to induce apoptosis is not restricted to cell type because DTMUV-induced apoptosis in duck and mammalian host cells. We further investigated which viral components induce apoptosis in DTMUV-infected host cells. The major envelope glycoprotein (E) was investigated for its apoptotic activities in expressed cells. Transient expression of the E protein alone triggered apoptosis in DEF, Vero, and BHK cells. Expression of the E protein resulted in activation of caspase-3-like proteases in cultured cells. These results indicate that infection of cells with DTMUV or expression of DTMUV E protein alone induces apoptosis, providing the basis for future to define the molecules that play key roles in the fate of DTMUV-infected cells.

Redistribution of subcellular calcium and its effect on apoptosis in primary cultures of rat proximal tubular cells exposed to lead.

Previous studies have shown that cytosolic Ca(2+) ([Ca(2+)]c) overload was involved in Pb-induced apoptosis in primary cultures of rat proximal tubular (rPT) cells, but the source of elevated Ca(2+) and the effect of potential subcellular Ca(2+) redistribution on apoptosis are still unknown. In this study, variations of [Ca(2+)]c in two culture media (Ca(2+)-containing and Ca(2+)- free) were analyzed, indicating that Pb-induced elevation of [Ca(2+)]c was primarily generated intracellularly. Fluo-4-AM, dihydro-Rhod-2-AM and Mag-Fluo-4-AM was loaded to Pb-exposed rPT cells to monitor the imaging of Ca(2+) concentrations in the cytoplasm ([Ca(2+)]c), mitochondria ([Ca(2+)]mit) and endoplasmic reticulum (ER) ([Ca(2+)]ER), respectively, under the confocal microscope. Data indicate that elevations of [Ca(2+)]c and [Ca(2+)]mit with depletion of [Ca(2+)]ER were revealed in Pb-treated rPT cells, but this subcellular Ca(2+) redistribution could be significantly suppressed by 2-APB, a specific inhibitor of inositol 1,4,5-trisphosphate receptor (IP3R) that functions to release Ca(2+) from ER stores. Simultaneously, Pb-mediated mitochondrial Ca(2+) overload can be partially suppressed by the cytosolic Ca(2+) chelator BAPTA-AM, suggesting that Ca(2+) uptake into mitochondria occurs via diverse pathways and ER Ca(2+) storage was the chief source. Furthermore, Pb-induced apoptosis was markedly inhibited by 2-APB and BAPTA-AM, respectively. Additionally, elevated IP3 levels with up-regulated IP3R-1 and IP3R-2 (mRNA and protein) levels were revealed in Pb-exposed rPT cells. In summary, IP3R-mediated ER Ca(2+) release promoted the elevations of [Ca(2+)]c and [Ca(2+)]mit in Pb-exposed rPT cells, which played a chief role in apoptosis induced by impaired calcium homeostasis.

Neuroprotective Mechanisms of 9-Hydroxy Epinootkatol Against Glutamate-Induced Neuronal Apoptosis in Primary Neuron Culture.

Glutamate-induced neuronal apoptosis has been implicated in the pathogenesis of neurological disorders. 9-Hydroxy epinootkatol (9OHEN), a sesquiterpene compound with neuroprotective activity against glutamate-induced neuronal apoptosis, was isolated from Alpinia oxyphylla Miquel in our previous study. In this study, we investigated the neuroprotective mechanisms of 9OHEN against glutamate-induced neuronal apoptosis in primary cultured neurons. The results from this study demonstrated that 9OHEN protected cortical neurons from glutamate-induced neuronal apoptosis via inhibiting glutamate-induced activation of caspase-3, inhibiting glutamate-induced reactive oxygen species (ROS) production, inhibiting glutamate-induced nitric oxide (NO) production, and downregulating glutamate-induced neuronal nitric oxide synthase (nNOS) expression. This study suggest that 9OHEN might have therapeutic potential in treating glutamate-mediated neurological diseases.

Correction: Epoxypukalide Induces Proliferation and Protects against Cytokine-Mediated Apoptosis in Primary Cultures of Pancreatic β-Cells

Correction: Epoxypukalide Induces Proliferation and Protects against Cytokine-Mediated Apoptosis in Primary Cultures of Pancreatic β-Cells

Epoxypukalide Induces Proliferation and Protects against Cytokine-Mediated Apoptosis in Primary Cultures of Pancreatic β-Cells

There is an urgency to find new treatments for the devastating epidemic of diabetes. Pancreatic β-cells viability and function are impaired in the two most common forms of diabetes, type 1 and type 2. Regeneration of pancreatic β-cells has been proposed as a potential therapy for diabetes. In a preliminary study, we screened a collection of marine products for β-cell proliferation. One unique compound (epoxypukalide) showed capability to induce β-cell replication in the cell line INS1 832/13 and in primary rat cell cultures. Epoxypukalide was used to study β-cell proliferation by [3H]thymidine incorporation and BrdU incorporation followed by BrdU/insulin staining in primary cultures of rat islets. AKT and ERK1/2 signalling pathways were analyzed. Cell cycle activators, cyclin D2 and cyclin E, were detected by western-blot. Apoptosis was studied by TUNEL and cleaved caspase 3. β-cell function was measured by glucose-stimulated insulin secretion. Epoxypukalide induced 2.5-fold increase in β-cell proliferation; this effect was mediated by activation of ERK1/2 signalling pathway and upregulation of the cell cycle activators, cyclin D2 and cyclin E. Interestingly, epoxypukalide showed protection from basal (40% lower versus control) and cytokine-induced apoptosis (80% lower versus control). Finally, epoxypukalide did not impair β-cell function when measured by glucose-stimulated insulin secretion. In conclusion, epoxypukalide induces β-cell proliferation and protects against basal and cytokine-mediated β-cell death in primary cultures of rat islets. These findings may be translated into new treatments for diabetes.

Yessotoxin as a Tool to Study Induction of Multiple Cell Death Pathways

This work proposes to use the marine algal toxin yessotoxin (YTX) to establish reference model experiments to explore medically valuable effects from induction of multiple cell death pathways. YTX is one of few toxins reported to make such induction. It is a small molecule compound which at low concentrations can induce apoptosis in primary cultures, many types of cells and cell lines. It can also induce a non-apoptotic form of programmed cell death in BC3H1 myoblast cell lines. The present contribution reviews arguments that this type of induction may have principal interest outside this particular example. One principal effect of medical interest may be that cancer cells will not so easily adapt to the synergistic effects from induction of more than one death pathway as compared to induction of only apoptosis.

Müllerian Inhibiting Substance Induces Apoptosis of Human Endometrial Stromal Cells in Endometriosis

Müllerian inhibiting substance (MIS) is produced in Sertoli cells of fetal testis and causes regression of müllerian ducts in male embryos. MIS also can induce the cell cycle arrest and apoptosis in müllerian duct-derived tumors in vivo and in vitro. Our objective was to investigate the expression of MIS type II receptor (MISR II) and whether MIS can inhibit the proliferation and induce apoptosis in primary cultures of endometrial stromal cells (ESC) of endometriosis. In vitro experiments were performed in the university research laboratory. Tissue samples from 12 patients who had undergone evisceration for ovarian endometrial cysts were included in this study. The expression of MISR II in ESC was investigated by immunohistochemistry. The cell viability and apoptosis in ESC treated with MIS was measured by methylthiazoletetrazolium assay and annexin V analysis. The expression of regulatory proteins in ESC treated with MIS was shown by Western blotting. ESC showed specific immunostaining for the MISR II. ESC treated with MIS exhibited 32% growth inhibition (P = 0.0001). The changes in cell cycle distribution after MIS exposure at 72 h demonstrated that S and G(2)M phases were decreased; G(0)G(1) and sub-G(0)G(1) phases were increased. ESC treated with MIS showed 13.72% annexin V-fluorescein isothiocyanate positivity. In the ESCs, which contain defective p16, MIS increased the expression of pocket proteins p107 and p130 and decreased E2F transcription factor 1. The results support a central role for MIS in endometriosis. Although the precise mechanism of MIS-mediated inhibition of ESC growth has not been fully defined, these data suggest that MIS has activity against ESC in vitro and may also be an effective targeted therapy for endometriosis.

Neurodegeneration and functional impairments associated with glycogen synthase accumulation in a mouse model of Lafora disease

Lafora disease (LD) is caused by mutations in either the laforin or malin gene. The hallmark of the disease is the accumulation of polyglucosan inclusions called Lafora Bodies (LBs). Malin knockout (KO) mice present polyglucosan accumulations in several brain areas, as do patients of LD. These structures are abundant in the cerebellum and hippocampus. Here, we report a large increase in glycogen synthase (GS) in these mice, in which the enzyme accumulates in LBs. Our study focused on the hippocampus where, under physiological conditions, astrocytes and parvalbumin-positive (PV+) interneurons expressed GS and malin. Although LBs have been described only in neurons, we found this polyglucosan accumulation in the astrocytes of the KO mice. They also had LBs in the soma and some processes of PV+ interneurons. This phenomenon was accompanied by the progressive loss of these neuronal cells and, importantly, neurophysiological alterations potentially related to impairment of hippocampal function. Our results emphasize the relevance of the laforin–malin complex in the control of glycogen metabolism and highlight altered glycogen accumulation as a key contributor to neurodegeneration in LD.

Lidocaine‐induced apoptosis of gingival fibroblasts: participation of cAMP and PKC activity

Local anaesthetics are drugs that prevent or relieve pain by interrupting nervous conduction and are the most commonly used drugs in dentistry. Their main targets of action are voltage-dependent Na+ channels. The Na+ channel is modulated by phosphorylation of two enzymes: PKA (protein kinase A) and PKC (protein kinase C). We studied the ability of lidocaine to modulate programmed cell death of human gingival fibroblasts and the mechanisms involved in this process. Lidocaine (10-5 to 10-7 M) stimulated apoptosis in primary cultures and the caspase-3 activity in a concentration-dependent manner. The stimulatory effect of lidocaine on apoptosis was attenuated in the presence of HA 1004 (PKA inhibitor) and stimulated by staurosporine and Go 6976 (PKC inhibitors). Lidocaine-induced apoptotic nuclei correlated positively with cAMP accumulation and negatively with PKC activity. These results show that lidocaine promotes apoptosis in human gingival fibroblasts at concentrations used for local anaesthesia. The mechanism involves PKA stimulation and PKC inhibition, which in turn stimulates caspase-3 and leads to programmed cell death.