Effects of Idebenone on Rat Schwann Cells with Toxicity Induced by Hydrogen Peroxide: Assessment of Molecular, Apoptosis, and Oxidative Stress Parameters.

OBJECTIVE: Schwann cells are the main cells for myelination and regeneration of peripheral nerves. Idebenone is a synthetic antioxidant used to treat central nervous system diseases. The aim of the study is to determine whether idebenone can protect Schwann cells and increase cell activity under conditions of oxidative stress caused by hydrogen peroxide (H2O2) in vitro. MATERIALS AND METHODS: In this experimental study, Schwann cells were pre-treated with various concentrations of idebenone and H2O2; after determining the appropriate doses, the cells were treated with 10 µM idebenone for 48 hours and 1000 µM H2O2 for the last two hours. The malondialdehyde (MDA) level, and activity of superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx) were assessed by ELISA. Cell viability was assessed by the MTT assay. Western blot analysis was conducted to determine the expressions of myelin protein zero (MPZ) and peripheral myelin protein 22 (PMP22), and expression ratio of the Bax/Bcl-2 proteins. The percentage of cell apoptosis was evaluated by annexin V staining using flow cytometry. RESULTS: Schwann cells under oxidative stress conditions caused by H2O2 and treated with idebenone had increased cell viability; increased SOD, CAT, and GPx activity; and increased expressions of the MPZ and PMP22 proteins. There was a decreased level of MDA, decreased expression ratio of Bax/Bcl-2 proteins, and a decrease in the percentage of apoptotic cells stained with Annexin V. CONCLUSION: The appropriate dose of idebenone may improve both survival and function of Schwann cells exposed to H2O2 by reducing oxidative stress and apoptosis.

Bone marrow stromal cell-conditioned medium regenerates injured sciatic nerve by increasing expression of MPZ and NGF and decreasing apoptosis.

Objectives: Despite the many benefits of mesenchymal stem cell (MSC) transplantation for tissue regeneration, there are some limitations to using them, including the high costs, applying invasive procedures, the possibility of transplant rejection, and cell malignancy. This study aimed to investigate the effect of secretions of bone marrow stromal cells (BMSCs) with the cell-free strategy on damaged sciatic nerve with an emphasis on the role of apoptosis and the expression of myelin protein zero (MPZ) and nerve growth factor (NGF) proteins. Materials and Methods: BMSCs were cultured and a 25-fold concentrated conditioned medium (CM) from the cells was provided. After creating a crush injury in the left sciatic nerve of male rats, BMSCs or CM were injected into the injured site of the nerve. Four weeks later, the expression of MPZ, NGF, Bax, and Bcl-2 proteins in the sciatic nerve and histological parameters of the sciatic nerve and gastrocnemius muscle were assessed. Results: The results demonstrated that injection of CM decreased apoptosis and increased expression of MPZ and NGF proteins, improving remyelination and regeneration of the sciatic nerve almost as much as the transplantation of the BMSCs themselves compared to the control group. Conclusion: The results suggest that BMSC secretions may improve remyelination and regeneration of damaged sciatic nerve by increasing the expression of MPZ and NGF and decreasing apoptosis.

Upregulation of Connexins in the Rat Hippocampal and Cortical Neurons Following Blockade of NMDA Receptors During Postnatal Development.

BACKGROUND: Interneural gap junctional coupling represents neural development that decreases during the postnatal period. The decrease of gap junction function coincides with the main period of chemical synapse creation and increment of synaptic activity during postnatal weeks 1 to 3. METHODS: Here, we have assessed the role of chemical synapses on connexin (Cx) expression in neurons and glial cells of hippocampal and cortical neurons. We characterized the impact of NMDA receptors blockade on the expression of Cx36 and Cx43 proteins by western blot analysis in postnatal day (PND)14 and PND28. MK801 was injected subcutaneously from the first day of birth until 14 or 28 days, depending on the experimental groups. Saline was injected in the same volumes in the control group. RESULTS: Early postnatal blockade of the NMDA subtype of glutamate receptors by the non-competitive antagonist dizocilpine maleate (MK801) arrested the developmental reduction in gap junctions during the initial postnatal weeks. Expression of Cx43 declined in PND28 compared to PND14 in visual cortex (VC) neurons. Also, we found that the expression of Cx36 and Cx43 augmented in the rats' VC in PND28 following the blockade of NMDA receptors. Expression of Cx36 declined in PND28 compared to PND14 in hippocampal neurons. Also, we found that the expression of Cx36 augmented in the rats' hippocampal neurons in PND14 and PND28 following a blockade of NMDA receptors. CONCLUSION: These results suggest that the postnatal enhancement in glutamatergic synaptic activity is associated with the loss of gap junctional connections and downregulation of Cx36 and Cx43 between developing neurons and glial cells.

Effect of sesamol on damaged peripheral nerves: Evaluation of functional, histological, molecular, and oxidative stress parameters.

Objective: Peripheral nerve injury is a clinical problem that may cause sensory and motor inabilities. Sesamol is an antioxidant that can help in repairing damaged central nervous system (CNS) and other organs. The present study aimed to investigate whether the antioxidant effects of sesamol could improve the function, structure, and myelination in rats' damaged peripheral nervous system (PNS). Materials and Methods: In this study, 28 adult male Wistar rats were randomly divided into four groups. In the sham group, the sciatic nerve was exposed and restored to its place without inducing crush injury. The control received DMSO (solvent) and the two experimental groups received 50 or 100 mg/kg sesamol intraperitoneally for 28 days after sciatic nerve crush injury, respectively. Next, sciatic function index (SFI), superoxide dismutase (SOD) activity, malondialdehyde (MDA) level, expression of nerve growth factor (NGF) and myelin protein zero (MPZ) proteins in the sciatic nerve, and histological indices of the sciatic nerve and gastrocnemius muscle were evaluated. Results: The results showed that sesamol reduced oxidative stress parameters, increased expression of NGF and MPZ proteins, and improved function and regeneration of the damaged sciatic nerve. Furthermore, a significant regeneration was observed in the gastrocnemius muscle after treatment with sesamol. Although administration of both doses of sesamol was useful, the 100 mg/kg dose was more effective than the 50 mg/kg one. Conclusion: The results suggest that sesamol may be effective in improving damaged peripheral nerves in rats by reducing oxidative stress and increasing the expression of NGF and MPZ proteins.

Sesamol protects the function and structure of rat ovaries against side effects of cyclophosphamide by decreasing oxidative stress and apoptosis.

AIM: Chemotherapy with cyclophosphamide can damage ovaries and cause infertility in girls and women. Sesamol is a phenolic antioxidant that can protect various organs from damage. The purpose of this study was to evaluate the effects of sesamol on protecting the function and structure of rat ovaries against the side effects of a chemotherapy model with cyclophosphamide. METHODS: Twenty-four adult female Wistar rats were randomly divided into three groups: (1) normal group, without any treatment, (2) control group, immediately after receiving cyclophosphamide, 0.5% dimethyl sulfoxide (DMSO) as the solvent of sesamol was intraperitoneally injected for 14 consecutive days, (3) sesamol group, immediately after receiving cyclophosphamide, 50 mg/kg sesamol was intraperitoneally injected for 14 consecutive days. Four weeks after the last injection, superoxide dismutase (SOD) activity and malondialdehyde (MDA) levels in the ovary, anti-Mullerian hormone (AMH) levels in the serum, number of ovarian follicles in different stages, and expression of proteins growth differentiation factor-9 (GDF-9), Bcl-2, and Bax in the ovary were evaluated. RESULTS: The results of SOD activity and MDA levels in the ovary, AMH levels in the serum, number of ovarian follicles in different stages, and expression of proteins GDF9, Bcl-2, and Bax in the ovary were significantly more favorable in the sesamol group than the control group. CONCLUSIONS: The results suggest that sesamol may protect function and structure in the rat ovaries against side effects of the chemotherapy model with cyclophosphamide by decreasing oxidative stress and apoptosis in the ovary.

Effects of Co-Administration of Bone Marrow Stromal Cells and L-Carnitine on The Recovery of Damaged Ovaries by Performing Chemotherapy Model in Rat.

BACKGROUND: L-carnitine (Lc) as a type of flavonoid antioxidants and bone marrow stromal cells (BMSCs) as a type of mesenchymal stem cells may recover damaged ovaries. It seems that Lc has favorable effects on differentiation, increasing lifespan and decreasing apoptosis in BMSCs. The aim of this study was to investigate effects of co-administration of BMSC+Lc on damaged ovaries after creating a chemotherapy model with cyclophosphamide in rats. MATERIALS AND METHODS: In this experimental study, cyclophosphamide was intraperitoneally (IP) injected to forty female wistar rats for 14 days, in terms of chemotherapy-induced ovarian destruction. The rats were then randomly divided into four groups: control, Lc, BMSCs and co-administration of BMSC+Lc. Injection of BMSCs into bilateral ovaries and intraperitoneal injection of Lc were performed individually and together. Four weeks later, levels of serum estradiol (E2) and follicle-stimulating hormone (FSH) using enzyme-linked immunosorbent assay (ELISA) kit, number of ovarian follicles at different stages using hematoxylin and eosin (H and E) staining and expression of ovarian Bcl-2 and Bax proteins using western blot were assessed. RESULTS: Co-administration of BMSC+Lc increased E2 and decreased FSH levels compared to the control group (P<0.001). The number of follicles was higher in the co-administrated group compared to the control group (P<0.001). Co-administration of BMSC+Lc increased Bcl-2 protein level, decreased Bax protein level and increased Bcl-2/Bax ratio (P<0.001). CONCLUSION: The effect of co-administration of BMSC+Lc is probably more effective than the effect of their separate administration on the recovery of damaged ovaries by chemotherapy.

Strong binding active constituents of phytochemical to BMPR1A promote bone regeneration: In vitro, in silico docking, and in vivo studies.

Two of the most problematic orthopedic and neurosurgeon visits are associated with spine and craniofacial fractures. Therefore, more attention needs to be paid to finding a medicine to repair these fractures. Amongst the most mysterious herbs, Aloe vera stands out. In the present study, the ameliorating function of A. vera on osteogenesis was studied in vitro and in vivo. Osteoblast-like cells were exposed to A. vera, followed by analysis of cell viability, lactate dehydrogenase release, and intracellular reactive oxygen species (ROS) production. The results showed an enhanced cell biocompatibility in a dose-dependent manner due to attenuated intracellular ROS production. Furthermore, a docking study indicated that the strong affinity of A. vera constituents to type I bone morphogenic protein receptor (BMPR1A) without the involvement of the BMPR1A chain B. The induction of osteogenesis prompts extracellular calcium deposition by osteoblasts, which affirms successful in vitro bone regeneration. However, injection of A. vera in rats with critical size calvarial defects induced Runx2, alkaline phosphatase (ALP), OCN, and BMP2 genes overexpression, which led to the formation of victorious bone with enhanced bone density and ALP activity. It is worthy to note that Aloin has the highest affinity to BMPR1A, whereas there are no reports regarding the impact of Aloenin, Aloesin, and γ-sitosterol on osteogenesis. Furthermore, some of them have antitumor potency, and it might be proposed that they are considered as a bone substitute in the osteotomy site of osteosarcoma with the aim of bone recovery and suppression of osteosarcoma. The whole consequences of this investigation manifests the plausibility of using A. vera as an antioxidant and osteoconductive substitute.

The effects of letrozole and clomiphene citrate on ligands expression of Wnt3, Wnt7a, and Wnt8b in proliferative endometrium of women with Polycystic ovarian syndrome.

Polycystic ovarian syndrome (PCOS) is a common endocrinologic disorder in women of reproductive age characterized by polycystic ovaries, oligo/anovulation, and hyperandrogenism. Not only anovulation but also endometrial dysfunction can reduce fertility in PCOS patients. Wnt pathway is responsible for endometrial proliferation which be strongly regulated by estradiol. To determine the effects of clomiphene citrate (CC) and letrozole, we measured the expression of some main ligands of Wnt/ß-catenin signaling including Wnt7a, Wnt3, and Wnt8b in the endometrial samples taken from PCOS women on day 12 of the menses who received 100 mg CC or 5 mg letrozole as well as from women without treatment. Significantly, the mean estrogen and progesterone concentration were lower and higher, respectively, in letrozole than CC. The mean endometrial thickness (ET) was significantly greater in letrozole compared to CC. Assessment of the mRNA and protein expression of Wnt7a, Wnt3, and Wnt8b showed significantly lower expression in CC than the letrozole and control groups. Collectively, letrozole provided a better molecular response in the endometrium of PCOS patients during the proliferative phase, similar to natural cycles, compared to CC. CC decreased the ligands expression of Wnt3, Wnt7a, and Wnt8b, resulting in endometrial dysfunction.

Swimming Training Attenuates Allodynia and Hyperalgesia Induced by Peripheral Nerve Injury in an Adult Male Rat Neuropathic Model: Effects on Irisin and GAD65.

Objective: The analgesic mechanism of long-lasting exercise on neuropathic pain is not well understood. This study explored the effects of swimming training on neuropathic pain and the expression of irisin, GAD65, and P2X3 after chronic constriction injury (CCI) of the sciatic nerve. Methods: Thirty-five male rats were randomly assigned to one of the following five groups: 1) no CCI or swimming (control); 2) swimming without CCI (SW); 3) swimming with CCI (CCISW); 4) CCI without swimming (CCI); and 5) sham CCI surgery (sham CCI). Behavioral responses to mechanical, cold, and heat stimuli were tested before and after CCI surgery, as well as each week throughout the four weeks of swimming training. The expression of irisin, GAD65, and P2X3 proteins in L4-L6 spinal cord segment, ipsilateral to the nerve injury, were evaluated by western blotting. Results: Mechanical hyperalgesia was alleviated between the second and fourth weeks of training in the CCISW group. In the tactile allodynia and heat hyperalgesia tests, withdrawal thresholds of the CCISW group were significantly higher than the CCI group at the third and fourth week of training (P < 0.05), while cold allodynia showed delayed improvement occurring by the fourth week of training. The expression of irisin was lower in the CCISW and SW groups compared with the CCI group at day 33 post-CCI surgery. Moreover, CCI surgery significantly decreased the protein expression of GAD65 in L4-L6 spinal cord segments (P = 0.018), whereas swimming training prevented the decline of GAD65 in the CCISW group. Conclusions: Our findings showed that four weeks of swimming training produce beneficial rehabilitative effects on neuropathic pain symptoms. The analgesic effect of swimming training is partially related to the increase of GAD65. The beneficial role of irisin in neuropathic pain will require further investigation.

Inhibition of DNA-PK enhances chemosensitivity of B-cell precursor acute lymphoblastic leukemia cells to doxorubicin.

DNA damage repair pathways greatly affect the response to genotoxic drugs in cancer cells, so inhibition of such pathways could be a potentially useful strategy to enhance chemosensitivity. DNA-dependent protein kinase (DNA-PK) plays a crucial role in the repair of DNA double-strand breaks (DSBs) that are probably one of the most detrimental types of DNA damage. It has been shown that DNA-PK is highly expressed in B-cell precursor acute lymphoblastic leukemia (BCP-ALL) cells. Less well appreciated was the effect of DNA-PK inhibition on sensitivity of BCP-ALL cells to DNA-damaging agents. Here, we show that the DNA-PK inhibitor NU7441 increased doxorubicin-induced apoptosis in BCP-ALL cell lines (NALM-6, SUP-B15), correlating with a reduction in DSB repair measured by γ-H2AX foci. NU7441 affected the cell cycle distribution and the cell cycle regulatory molecules in combination with doxorubicin treatment. Doxorubicin-induced DNA-PK phosphorylation was decreased in the presence of NU7441. Apoptosis induction by the combined treatment was associated with marked reduction of Bcl-2 and survivin and a significant increase of Bax mRNA expression levels. In conclusion, our data indicate that inhibition of DNA-PK might be an effective approach to enhance the tumor-cell-killing effects of DNA-damaging agents such as doxorubicin in BCP-ALL and may deliver novel, targeted therapy into the clinic.

Protective mechanisms of melatonin against hydrogen-peroxide-induced toxicity in human bone-marrow-derived mesenchymal stem cells.

Many obstacles compromise the efficacy of bone marrow mesenchymal stem cells (BM-MSCs) by inducing apoptosis in the grafted BM-MSCs. The current study investigates the effect of melatonin on important mediators involved in survival of BM-MSCs in hydrogen peroxide (H2O2) apoptosis model. In brief, BM-MSCs were isolated, treated with melatonin, and then exposed to H2O2. Their viability was assessed by MTT assay and apoptotic fractions were evaluated through Annexin V, Hoechst staining, and ADP/ATP ratio. Oxidative stress biomarkers including ROS, total antioxidant power (TAP), superoxide dismutase (SOD) and catalase (CAT) activity, glutathione (GSH), thiol molecules, and lipid peroxidation (LPO) levels were determined. Secretion of inflammatory cytokines (TNF-α and IL-6) were measured by ELISA assay. The protein expression of caspase-3, Bax, and Bcl-2, was also evaluated by Western blotting. Melatonin pretreatment significantly increased viability and decreased apoptotic fraction of H2O2-exposed BM-MSCs. Melatonin also decreased ROS generation, as well as increasing the activity of SOD and CAT enzymes and GSH content. Secretion of inflammatory cytokines in H2O2-exposed cells was also reduced by melatonin. Expression of caspase-3 and Bax proteins in H2O2-exposed cells was diminished by melatonin pretreatment. The findings suggest that melatonin may be an effective protective agent against H2O2-induced oxidative stress and apoptosis in MSC.

Rolipram potentiates bevacizumab-induced cell death in human glioblastoma stem-like cells.

AIMS: Glioblastoma cancer stem-like cells (GCSCs) promote themselves proliferation by secreting the vascular endothelial growth factor A (VEGFA) in an autocrine manner, positively regulated by phosphodiesterase IV (PDE4). In the current study, we investigated the putative cytotoxic effect of bevacizumab, a VEGFA blocker, alone and in combination with a specific inhibitor of PDE4 called rolipram on GCSCs isolated from human surgical tumor specimen with a focus on PI3K/AKT pathway. MAIN METHODS: CD133+/CD15+ GCSCs were characterized by flow cytometry and expanded in a serum-free primary culture system. The cell survival, apoptosis, and protein expression values were measured using MTT assay, TUNEL staining and western blot, successively. Intracellular cAMP and free secreted VEGFA levels were assessed by cAMP enzyme immunoassay and ELISA, respectively. KEY FINDINGS: Bevacizumab suppressed GCSCs survival with IC50~6.5µg/ml and enhanced the levels of apoptosis, p53 and cleaved-caspase3 along with a decrease in free VEGFA levels and ERKs activation. However, there was no significant modulation of AKT phosphorylation on serine 473, the intracellular PDE4A, VEGFA and cAMP levels. More cytotoxicity in co-treated cells coupled with a more substantial decline in the free VEGFA levels and a greater increase in the quantities of p53 and cleaved-caspase3 compared to those treated with bevacizumab alone. Co-treatment reduced phospho-AKT, endogenous VEGFA and PDE4A values but elevated cAMP levels. SIGNIFICANCE: This study highlighted a booster cytotoxic effect of combined rolipram and bevacizumab treatment on the GCSCs primary culture, suggesting that this approach is warranted in treatment of GBMs overexpressing VEGFA and PDE4A.

Photobiomodulation therapy reduces apoptotic factors and increases glutathione levels in a neuropathic pain model.

Neuropathic pain (NP) is caused by damage to the nervous system due to reactive oxygen spices (ROS) increase, antioxidants reduction, ATP production imbalance, and induction of apoptosis. In this investigation, we applied low-level laser 660 nm (photobiomodulation therapy) as a new strategy to modulate pain. In order to study the effects of photobiomodulation therapy (660 nm) on NP, chronic constriction injury (CCI) model was selected. Low-level laser of 660 nm was used for 2 weeks. Thermal and mechanical hyperalgesia were measured before and after surgery on days 7 and 14, respectively. Paw withdrawal thresholds were also evaluated. Expression of p2x3, Bax, and bcl2 protein was measured by western blotting. The amount of glutathione (GSH) was measured in the spinal cord by continuous spectrophotometric rate determination method. The results are presented as mean ± SD. Statistical analysis of data was carried out using SPSS 21. CCI decreased the pain threshold, 2-week photobiomodulation therapy significantly increased mechanical and thermal threshold, decreased P2X3 expression (p < 0.001), and increased bcl2 expression (p < 0.01), but it was not effective on the Bax expression. We speculated that although photobiomodulation therapy increased ROS generation, it increased antioxidants such as GSH. Increase in bcl2 is another mitochondrial protection mechanism for cell survival and that pain relief and decrease in P2X3 expression confirm it.

Induction of endothelial cell proliferation and von Willebrand factor expression and secretion by leukemic plasma of patients with chronic lymphocytic leukemia before and after inhibition of NF-κB.

Although certain evidence has indicated a role for angiogenesis in the pathophysiology of hematopoietic malignancies, its role in chronic lymphocytic leukemia (CLL) prognosis is yet to be defined. To our knowledge, the effects of CLL plasma on cell culture have not been addressed. Therefore, we investigated the effects of CLL plasma on cell cycle regulation and von Willebrand factor (vWF) secretion, and expression in human umbilical vein endothelial cell cultures (HUVECs). Since nuclear factor-kappa B (NF-κB) transcription factor has been a therapeutic target for treatment of cancer, we inhibited NF-κB using small interfering RNA to clarify if there is a role for this factor in probable effects. The cells were treated with the plasma of patients with CLL. Subsequently, cell cycle phase distribution, vWF secretion, expression, and storage were detected using ELISA, flow cytometry, and immunohistochemical staining. In addition, NF-κB was inhibited using small interfering RNA. Plasma treatment promoted cell cycle progression by decreasing the cell number in G1 phase, while increasing the cell number in S phase and G2M phase. A significant increase of vWF expression, secretion, and storage was found, associated with the vWF levels of patients' plasma. We found that induction of cell cycle promotion, but not vWF expression and secretion, was partially suppressed by this inhibition. We found that endothelial cell cycle and vWF expression and secretion affected by CLL plasma and NF-κB play a role in the former. These findings would be useful for understanding the prognostic importance of plasma angiogenic factor levels in CLL.

BDNF modifies hippocampal KCC2 and NKCC1 expression in a temporal lobe epilepsy model.

Excitatory GABA actions, induced by altered expression of chloride transporters (KCC2/NKCC1), can contribute to seizure generation in temporal lobe epilepsy. In the present study, we evaluated whether BDNF administration can affect KCC2/NKCC1 expression, ictogenesis and behavioral alterations in this paradigm. Status epilepticus was induced in male rats with pilocarpine, followed by a treatment of either a single high dose or multiple injections of BDNF during the latent phase of temporal lobe epilepsy. Chloride transporters expression, spontaneous recurrent seizures, and hyperexcitability post-seizural behaviors were evaluated after treatment. NKCC1 protein expression was markedly upregulated, whereas that of KCC2 was significantly downregulated in epileptic hippocampi compared to intact controls. Application of BDNF (both single high dose and multiple injections) increased KCC2 expression in epileptic hippocampi, while NKCC1 expression was downregulated exclusively by the single high dose injection of BDNF. Development of spontaneous recurrent seizures was delayed but not prevented by the treatment, and hyperexcitability behaviors were ameliorated for a short period of time. To prevent GABA-A mediated depolarization and design appropriate treatment strategies for temporal lobe epilepsy, chloride transporters can be considered as a target. Future studies are warranted to investigate any possible therapeutic effects of BDNF via altering chloride transporters expression.

The in vitro effects of sodium salicylate on von Willebrand factor and C-reactive protein production by endothelial cells.

OBJECTIVE: The role of inflammatory and endothelial dysfunction markers in the atherogenic process has been well recognized. The data have made both C-reactive protein (CRP) and von Willebrand factor (vWF) promising targets for the cardiovascular disease research and drug development. Inhibition of CRP and vWF synthesis, therefore, might be a potential therapeutic strategy. METHODS: The effect of sodium salicylate on vWF production by human umbilical vein endothelial cells (HUVECs) using enzyme-linked immunosorbent Assays (ELISA) and real-time PCR was examined. In addition, small interfering RNA (siRNA) against NF-κB was used to investigate the existence of a role for this signaling pathway. RESULTS: Our findings demonstrated that sodium salicylate decreased vWF, but not CRP production at both mRNA and protein levels significantly and this might not occur via nuclear transcription factor (NF-κB) inhibition. CONCLUSION: Our results indicated a further rationalization of the effects of sodium salicylate on atherothrombotic events by attenuation of vWF production.

Elevation of cyclic AMP causes an imbalance between NF-kappaB and p53 in NALM-6 cells treated by doxorubicin.

We previously showed that cAMP can inhibit DNA damage-induced wild type p53 accumulation in human pre-B NALM-6 cells, leading to a profound reduction of their apoptotic response. Here, we provide evidence for the potentiation of DNA damage-induced NF-kappaB activation by cAMP. We found that inhibition of NF-kappaB activation prevents the inhibitory effect of cAMP on doxorubicin-induced apoptosis. Moreover, cAMP exerts its inhibitory effect on doxorubicin-induced apoptosis in a PKA-independent manner. The present study also shows that elevation of cAMP prolongs the phosphorylation of IkappaB and subsequent activation of NF-kappaB in doxorubicin treated NALM-6 cells in a proteasome-dependent manner. Taken together, our results demonstrate that cAMP abrogates the balance between apoptotic and antiapoptotic transcription factors that are hallmarks of DNA damage signaling.

Inhibitory role of cAMP on doxorubicin-induced apoptosis in pre-B ALL cells through dephosphorylation of p53 serine residues.

Exposure of cells to chemotherapeutic drug doxorubicin, a DNA-damaging agent, induces an increase in the levels and activity of the wild-type p53 protein. Less well appreciated was the effect of cAMP levels on posttranslational modifications of p53 in response to doxorubicin. Here we show that elevation of cAMP in pre-B acute lymphoblastic leukemia NALM-6 cells significantly attenuated phosphorylation state of p53 at Ser6, Ser9, Ser15, Ser20, Ser37, Ser46 and Ser392 upon exposure to doxorubicin. Increased cAMP levels also shifted the ratio of the death promoter to death repressor genes via alteration of Bcl-2 and Bax proteins expression. In conclusion, our results suggest that activation of cAMP-signaling system may repress p53-dependent apoptosis in malignant cells exposed to doxorubicin.

Immediate exposure to TNF-alpha activate dendritic cells derived from non-purified cord blood mononuclear cells.

BACKGROUND: Tumor necrosis factor alpha (TNF-alpha) is a primary mediator of immune regulation and might be required in the early stages of DC development from CD34+ cells. However, details of optimal timing of exposure to TNF-alpha in DC development process in monocytes or non-purified hematopoitic cells are still lacking and clear benefits of this approach to the development of DCs remain to be validated. OBJECTIVE: To evaluate the effect of early and late exposure to TNF-alpha on DC development from non-purified cord blood mononuclear cells. METHODS: To define the effects of early exposure to TNF-alpha on cord blood mononuclear cells, we cultured UCB-MNC in the presence of SCF, Flt3L, GM-CSF and IL-4 for 14 days and matured them for an extra 4 days. TNF-alpha was added on day 0, 7 and 14 in TNF-alpha + group, and only on day 14 in TNF-alpha - group where it was used only as a maturation factor. RESULTS: Immediate exposure to TNF-alpha was shown to: (1) enhance the survival of cells in the first week of culture; (2) produce mature DCs with higher maturation markers (CD80, CD83, CD86 and HLA-DR); and (3) increase secretion of IL-12 by mature DCs. In contrast, delayed exposure to TNF-alpha stimulate mature DCs with less purity producing a high level of IL-10 and a low level of IL-12. CONCLUSION: We developed a simple, easy and cost effective method to generate DCs from non-fractionating mononuclear cells in this study. Also we confirm the presence of a large number of functional DCs under inflammatory conditions, where local concentrations of TNF-alpha were high.