Search

Diabetic peripheral neuropathy (DPN) is mainly characterized by demyelination resulted from the apoptosis of the Schwann cell (SCs). Although the exact mechanisms underlying DPN remain unclear, endoplasmic reticulum (ER) stress is strongly implicated in the apoptosis. Under ER stress, activated inositol-requiring kinase 1α (IRE1α) unregulated CHOP, phosphorylated JNK and Caspase-12 to aggravate apoptosis-mediated damage of DPN. Therefore, we tested the hypothesis that inhibition of IRE1α could reduce the ER stress-related apoptosis to relieve DPN. Here, we show that IRE1α siRNA improved the neurological morphology and function of DPN rats and rescued ER stress-related apoptosis in the sciatic nerve. Additionally, RSC96 cells transfected with IRE1α siRNA were used as in vitro model of DPN. It was found that IRE1α siRNA also decreased high glucose-induced apoptosis and inhibited ER stress-related apoptosis in the cells. Altogether, our results suggest that IRE1α should be considered a potential therapeutic agent for DPN.

In this study, in vitro and in vivo experiments were carried out with the high-affinity multifunctional D2/D3 agonist D-512 to explore its potential neuroprotective effects in models of Parkinson's disease and the potential mechanism(s) underlying such properties. Pre-treatment with D-512 in vitro was found to rescue rat adrenal Pheochromocytoma PC12 cells from toxicity induced by 6-hydroxydopamine administration in a dose-dependent manner. Neuroprotection was found to coincide with reductions in intracellular reactive oxygen species, lipid peroxidation, and DNA damage. In vivo, pre-treatment with 0.5 mg/kg D-512 was protective against neurodegenerative phenotypes associated with systemic administration of MPTP, including losses in striatal dopamine, reductions in numbers of DAergic neurons in the substantia nigra (SN), and locomotor dysfunction. These observations strongly suggest that the multifunctional drug D-512 may constitute a novel viable therapy for Parkinson's disease., (© 2014 International Society for Neurochemistry.)

Currently, unresectable esophageal squamous cell carcinoma (ESCC) is primarily treated by chemoradiotherapy. However, the outcome has not improved significantly because of radioresistance of cancer cells. This study aimed to determine the radiosensitizing effect of melittin, a novel component of bee venom, in ESCC. ESCC cell lines were irradiated with or without melittin. Cell proliferation was detected by Cell Counting Kit 8 assay. Radiosensitization was evaluated by clonogenic survival assay. Cell apoptosis was detected by flow cytometry. Results show that melittin potently sensitized ESCC cells to radiation with a sensitization enhancement ratio of 1.15-1.42. Radiosensitization was accompanied with enhanced apoptosis and regulated by apoptosis proteins. The results were confirmed by in vivo studies on tumor-bearing xenografts. In summary, these results provide support that melittin may be a potentially promising radiosensitizer in ESCC radiation therapy.

Triple negative breast cancer (TNBC) has increased recurrence and poor survival, despite a high response rate to neoadjuvant chemotherapy. The aim of this study was to determine whether current drug treatment(s) eliminates bulk of tumor cells, but it has a minimal effect on cancer stem cells (CSCs) leading to tumor recurrence. We studied the effects of PARP inhibitors (AZD2281 and BSI-201), paclitaxel, docetaxel, cisplatin and cisplatin plus TRAIL on CSCs derived from CRL-2335 and MDA-MB-468 TNBC cells in vitro. The in vitro data indicate that cisplatin plus TRIAL treatment was most effective in eliminating CSCs compared to PARP inhibitors, cisplatin, paclitaxel and docetaxel. Treatment with cisplatin plus TRAIL also inhibits Wnt-1 signaling and its downstream target, β-catenin, phospho β-catenin, cyclin D1, increased apoptosis, reduced proliferation and mammosphere formation. Inhibition of Wnt-1 by siRNA significantly reduced the ability of CSCs to form mammospheres compared to control. However, maximum effect was seen in cisplatin plus TRAIL-treated cells. Taken together the data suggest that cisplatin plus TRAIL treatment has the potential of providing a new strategy for improving the therapeutic outcome in TNBC patients.

The adamantyl-substituted retinoid-related (ARR) compounds 3-Cl-AHPC and AHP3 induce apoptosis in vitro and in vivo in a newly established human acute myelogenous leukemia (AML) cell line, FFMA-AML, and in the established TF(v-SRC) AML cell line. FFMA-AML and TF(v-SRC) cells displayed resistance to apoptosis mediated by the standard retinoids (including trans-retinoic acid, 9-cis-retinoic acid, and the synthetic retinoid TTNPB) but showed sensitivity to apoptosis mediated by 3-Cl-AHPC- and AHP3 in vitro and in vivo as documented by poly(ADP-ribose) polymerase (PARP) cleavage and apoptosis terminal deoxyribonucleotidyl transferase-mediated dUTP nick end labeling assay. 3-Cl-AHPC or AHP3 exposure in vitro resulted in decreased expression of the antiapoptotic proteins (cellular inhibitor of apoptosis 1, X-linked inhibitor of apoptosis protein) and phospho-Bad and activated the NF-κB canonical pathway. A significant prolongation of survival was observed both in nonobese diabetic severe combined immunodeficient mice carrying FFMA-AML cells and treated with either 3-Cl-AHPC or AHP3 and in severe combined immunodeficient mice carrying TF(v-SRC) cells and treated with AHP3. We have previously shown that ARRs bind to the orphan nuclear receptor small heterodimer partner (SHP) and that the expression of SHP is required for ARR-mediated apoptosis. Induced loss of SHP in these AML cells blocked 3-Cl-AHPC- and AHP3-mediated induction of apoptosis. These results support the further development of 3-Cl-AHPC and AHP3 as potential therapeutic agents in the treatment of AML patients., (©2010 AACR.)

We previously found that the adamantyl-substituted retinoid-related molecules bind to the small heterodimer partner (SHP) as well as the Sin3A complex. In this report, we delineated the role of SHP and the Sin3A complex in 4-[3'-(1-adamantyl)-4'-hydroxyphenyl]-3-chlorocinnamic acid (3-Cl-AHPC)-mediated inhibition of cell growth and apoptosis. We examined the effect of loss of SHP and Sin3A expression in a number of cell types on 3-Cl-AHPC-mediated growth inhibition and apoptosis induction, 3-Cl-AHPC-mediated nuclear factor-kappaB (NF-kappaB) activation, and 3-Cl-AHPC-mediated increase in c-Fos and c-Jun expression. We found that loss of SHP or Sin3A expression, while blocking 3-Cl-AHPC-mediated apoptosis, had little effect on 3-Cl-AHPC inhibition of cellular proliferation. We have previously shown that 3-Cl-AHPC-mediated NF-kappaB activation is necessary for apoptosis induction. We have now shown that 3-Cl-AHPC-enhanced c-Fos and c-Jun expression is also essential for maximal 3-Cl-AHPC-mediated apoptosis. 3-Cl-AHPC induction of c-Fos and c-Jun expression as well as NF-kappaB activation was dependent on SHP protein levels. In turn, SHP levels are regulated by Sin3A because ablation of Sin3A resulted in a decrease in SHP expression. Thus, SHP and Sin3A play an important role in adamantyl-substituted retinoid-related induction of cellular apoptosis.

The involvement and potential interdependence of inositol trisphosphate (IP3) receptors and Bcl-2 in the regulation of Ca2+ signaling is not clear. Here, we have explored the mechanism(s) of how Bcl-2 suppresses the IP3-sensitive Ca2+ release in MCF-7 cells focusing on the possible role of protein phosphatase 1 (PP1). We found that through influences on protein-protein interaction, Bcl-2 may alter the balance between the effects of phosphatase (PP1) and kinase (PKA) on the IP3 R1 signaling complex. Using various experimental approaches including phosphatase inhibition and RNAi, we show that Bcl-2 by competing with IP3R1 for the binding of PP1 can reduce the IP3-mediated calcium signal and protect cells from mitochondrial dysfunction and cell death.

Objectives: We have previously demonstrated that cocaine exposure leads to apoptosis in rat testes. To understand further the mechanism of cocaine-induced testicular damage, we studied the effect of cocaine on cytochrome c release from the mitochondria. We also determined the caspase 3, caspase 8, and caspase 9 activities in rat testes after chronic cocaine exposure., Methods: Thirty-day-old male Sprague-Dawley rats received cocaine hydrochloride or equal volumes of normal saline subcutaneously daily for 90 days. The testes were removed at 15, 30, and 90 days of cocaine or saline administration. Mitochondria and cytosolic fractions from testes were isolated. Western blotting was performed in both fractions using anti-cytochrome c antibody. Caspase 3, caspase 8, and caspase 9 activities were determined by fluorometric assay., Results: The expression of cytochrome c protein in the cytosolic fraction was increased on day 15 and persisted for up to 90 days after cocaine injection compared with controls. However, the expression of cytochrome c in testes was decreased in the mitochondria fraction on days 15, 30, and 90 after cocaine injections compared with the corresponding controls. The caspase activity study showed caspase 3 and caspase 9 activities increased in cocaine-treated testes at each point of the study compared with the corresponding controls. However, the caspase 8 activity in cocaine-treated testes did not change significantly at each point of the study compared with the corresponding controls., Conclusions: Our results suggest that the release of cytochrome c from mitochondria and its subsequent activation of caspase 9 and caspase 3 in testes play a key role in cocaine-induced germ cell apoptosis. Our findings also indicate that cocaine-induced testicular germ cell apoptosis in rats is at least initiated through a mitochondria-associated pathway.

Background. Diabetic peripheral neuropathy (DPN) is a common complication of diabetes but its pathogenesis is not fully clarified. Endoplasmic reticulum (ER) stress has been confirmed to be involved in the development of DPN. Dorsal root ganglion neuron (DRGn) is the target cell of DPN injure in the peripheral neurons system. Schwann cell (SCs)-derived exosomes (SC-EXOs) can carry IRE1α signal transduction factors in ER stress to DRGn. The aim of this study is to investigate the effect of SC-EXOs treated with paeoniflorin (PF) on DRGn stimulated by high glucose. Methods. SCs were divided into Control group (Control), 150 mM glucose group (HG), high osmotic pressure group (HOP), and low, middle, and high dose PF group (PF1, PF10, and PF100). Exosomes were obtained from SCs by ultracentrifugation and identified according to marker proteins, including CD63, Alix, Hsp70, and TSG101. ER stress initiating factor GRP78, the IRE1α pathway information transmission factor IRE1α, and the phosphorylation level of IRE1α were detected by Western blot, DRGn is divided into Control group (Control), 50 mM glucose group + Control exosomes group (HG + EXOs Control), 50 mM glucose group (HG), and 50 mM glucose group + administration exosomes group (HG + EXOs PF1, HG + EXOs PF10, and HG + EXOs PF100); ER morphology of primary DRGn was observed by using the transmission electron microscope, the level of DRGn apoptosis was analyzed by TUNEL, and the downstream proteins of ER stress including CHOP, XBP1S, JNK, and p-JNK in DRG and the expression of apoptosis-related proteins Bcl-2, Bax, Caspase-3, and Caspase-12 were measured by Western blot. Results. Compared with the exosomes in the HG group, the exosomes after the intervention of PF can significantly reduce the expression of GRP78, IRE1α, and the phosphorylation level of IRE1α P < 0.05 ; compared with the DRGn in the HG group, the SC-EXOs treated with PF could regulate the expression of proteins downstream of IRE1α pathway in ER stress (P < 0.05 or P < 0.01), improve the morphological integrity of ER, and reduce apoptosis in DRGn (P < 0.05 or P < 0.01). Conclusion. PF regulates the information of ER stress carried by SC-EXOs and further affects downstream of IRE1α pathway in DRGn, thus reducing ER stress-induced apoptosis. PF can interfere with DPN through affecting information communication carried by EXOs between SCs and DRGn. [ABSTRACT FROM AUTHOR]

Disease lesion mimic (Les / les) mutants display disease-like spontaneous lesions in the absence of pathogen infection, implying the constitutive activation of defense responses. However, the genetic and biochemical bases underlying the activated defense responses in those mutants remain largely unknown. Here, we performed integrated transcriptomics and metabolomics analysis on three typical maize Les mutants Les4 , Les10 , and Les17 with large, medium, and small lesion size, respectively, thereby dissecting the activated defense responses at the transcriptional and metabolomic level. A total of 1,714, 4,887, and 1,625 differentially expressed genes (DEGs) were identified in Les4 , Les10 , and Les17 , respectively. Among them, 570, 3,299, and 447 specific differentially expressed genes (SGs) were identified, implying a specific function of each LES gene. In addition, 480 common differentially expressed genes (CGs) and 42 common differentially accumulated metabolites (CMs) were identified in all Les mutants, suggesting the robust activation of shared signaling pathways. Intriguingly, substantial analysis of the CGs indicated that genes involved in the programmed cell death, defense responses, and phenylpropanoid and terpenoid biosynthesis were most commonly activated. Genes involved in photosynthetic biosynthesis, however, were generally repressed. Consistently, the dominant CMs identified were phenylpropanoids and flavonoids. In particular, lignin, the phenylpropanoid-based polymer, was significantly increased in all three mutants. These data collectively imply that transcriptional activation of defense-related gene expression; increase of phenylpropanoid, lignin, flavonoid, and terpenoid biosynthesis; and inhibition of photosynthesis are generalnatures associated with the lesion formation and constitutively activated defense responses in those mutants. Further studies on the identified SGs and CGs will shed new light on the function of each LES gene as well as the regulatory network of defense responses in maize. [ABSTRACT FROM AUTHOR]

Purpose: This study aimed to evaluate the regulatory role of miR-431-5p on the tumorigenesis of osteosarcoma (OS) and the underlying mechanism involving pannexin 3 (PANX3). Methods: qRT-PCR was applied to measure the expression of miR-431-5p in OS tissues and cells. PANX3 and miR-431-5p were overexpressed in U2OS and HOS cells. The cell viability and apoptosis were determined by MTT and FITC/PI double staining assay, respectively. Transwell assay was performed to detect cell migration and invasion. The protein expression of cleave-caspase-3 and MMP-2/-9 was detected by Western blot. The target relationship between miR-431-5p and PANX3 was predicated by ENCORI and identified by DLR assay. The anti-tumor effect of miR-431-5p was further analyzed in a xenograft tumor model in mice. Results: MiR-431-5p expression was down-regulated in OS tissues and negatively correlated with lymph node metastasis and TNM stage. Over-expression of miR-431-5p induced cell apoptosis, inhibited cell proliferation, migration and invasion, up-regulated cleave-caspase-3, and down-regulated MMP-2 and -9 in OS cells. Over-expression of miR-431-5p also inhibited the growth of tumor xenografts in mice. In addition, PANX3 was a target of miR-431-5p. Over-expression of PANX3 reversed the anti-tumor effect of miR-431-5p mimics on U2OS and HOS cells. Conclusion: Up-regulation of miR-431-5p suppressed the tumorigenesis of OS via targeting PANX3. [ABSTRACT FROM AUTHOR]

Radiotherapy is a useful treatment for malignant tumors, including lung carcinoma; however, non-small cell lung carcinoma (NSCLC) is frequently insensitive to radiation. It has been reported that heat shock protein 27 (HSPB1) is a radioresistance-associated protein in nasopharyngeal carcinoma. In the present study, the role of HSPB1 in NSCLC cells induced by irradiation was investigated. The viability of cells was determined by a Cell Counting Kit-8 assay. The apoptotic activity, cell cycle distribution and mitochondrial membrane potential (MMP) of cells were evaluated via flow cytometry. Reverse transcription-quantitative polymerase chain reaction and western blot analyses were employed to measure the expression of various genes and proteins. It was observed that knockdown of HSPB1 with small interfering RNA (si-HSPB1) markedly decreased the viability of A549 NSCLC cells and induced cell cycle arrest in the G2/M phase following exposure to 6 Gy irradiation. Furthermore, it was revealed that si-HSPB1 significantly downregulated cyclin B1 and cyclin G1 expression. Additionally, si-HSPB1 promoted apoptosis and depolarized the MMP of cells exposed to 6 Gy irradiation. The expression levels of B-cell lymphoma-2 (Bcl-2), mitochondrial cytochrome c (cyto c) and pro-caspase-8 were downregulated, whereas those of Bcl-2 associated X protein (Bax), cytosolic cyto c and cleaved-caspase-8 were upregulated. Collectively, silencing of HSPB1 increased the radiosensitivity of NSCLC cells by reducing cell viability, depolarizing the MMP, arresting the cell cycle in the G2/M phase and promoting cell apoptosis. Therefore, HSPB1 may be a novel target for increasing radiosensitivity in the treatment of NSCLC. [ABSTRACT FROM AUTHOR]

Tang-Luo-Ning (TLN) has a definite effect in the clinical treatment of diabetic peripheral neuropathy (DPN). Schwann cells (SCs) apoptosis induced by endoplasmic reticulum stress (ER stress) is one of the main pathogeneses of DPN. This study investigates whether TLN can inhibit SCs apoptosis by inhibiting ER stress-induced apoptosis. Our previous researches have demonstrated that TLN could increase the expression of ER stress marker protein GRP78 and inhibited the expression of apoptosis marker protein CHOP in ER stress. In this study, the results showed that TLN attenuated apoptosis by decreasing Ca2+ level in SCs and maintaining ER morphology. TLN could decrease downstream proteins of CHOP including GADD34 and Ero1α, while it increased P-eIF2α and decreased the upstream proteins of CHOP including P-IRE1α/IRE1α and XBP-1, thereby reducing ER stress-induced apoptosis. [ABSTRACT FROM AUTHOR]

Currently, unresectable esophageal squamous cell carcinoma (ESCC) is primarily treated by chemoradiotherapy. However, the outcome has not improved significantly because of radioresistance of cancer cells. This study aimed to determine the radiosensitizing effect of melittin, a novel component of bee venom, in ESCC. ESCC cell lines were irradiated with or without melittin. Cell proliferation was detected by Cell Counting Kit 8 assay. Radiosensitization was evaluated by clonogenic survival assay. Cell apoptosis was detected by flow cytometry. Results show that melittin potently sensitized ESCC cells to radiation with a sensitization enhancement ratio of 1.15-1.42. Radiosensitization was accompanied with enhanced apoptosis and regulated by apoptosis proteins. The results were confirmed by in vivo studies on tumor-bearing xenografts. In summary, these results provide support that melittin may be a potentially promising radiosensitizer in ESCC radiation therapy. [ABSTRACT FROM AUTHOR]

Objectives To investigate the biological function of eEF2K in esophageal squamous cell carcinoma (ESCC). Materials and methods Tissue microarrays containing 100 pairs of ESCC tumor and adjacent normal tissues were completed. Overexpression and knockdown of eEF2K were constructed in ECA-109 and TE-13 ESCC cells. DNA damage, cell viability, migration and invasion, radioresistance, apoptosis and autophagy were determined by immunofluorescence, CCK-8, transwell assay, colony formation assay, flow cytometry and western blot, respectively. Tumor growth and radioresistance were also evaluated using xenograft models created in nude mice. Results eEF2K expression was higher in ESCC tissues compared with matched non-tumor tissues ( P < 0.05). Proliferation was increased in eEF2K overexpressing cells compared with controls ( P < 0.05), while silencing eEF2K reduced cell proliferation ( P < 0.05). Furthermore, lower levels of eEF2K expression correlated with slower migration and invasion rates ( P < 0.05), while higher levels of eEF2K expression with faster migration and invasion rates ( P < 0.05). eEF2K overexpression resulted in radioresistance and radiation-induced autophagy, and reduced radiation-induced apoptosis compared with controls, but silencing eEF2K promoted radiosensitivity and apoptosis, and reduced autophagy. In addition, eEF2K overexpression promoted the tumor growth in vivo ( P < 0.01). Combined treatment of NH125 (a pharmacological inhibitor of eEF2K) and radiation was more effective at delaying xenograft tumor growth than NH125 and radiation alone ( P < 0.05). Conclusion eEF2K induced progression and radioresistance in ESCC, which may be a novel therapeutic target for ESCC to increase radiosensitivity. [ABSTRACT FROM AUTHOR]

Ethnopharmacological relevance Paeoniflorin (PF) is the principal bioactive component of Paeonia lactiflora Pall., which an included in Tang Luo Ning recipe, a traditional Chinese herbal medicine based on Huangqi Guizhi Wuwu decoction. PF is also widely used in Traditional Chinese Medicine for the treatment of blood-arthralgia disease including diabetic peripheral neuropathy (DPN), but its underlying molecular mechanism of neuroprotective effects is not yet well understood. Diabetic hyperglycemia induced oxidative stress in Schwann cells, an important component of the peripheral nervous system, has been proposed as a unifying mechanism for DPN. The objective of this study is to determine the effects of PF on Schwann cells oxidative stress and apoptosis induced by high glucose. Materials and methods RSC96 cells, a Schwann cell line, were treated with high glucose (150 mM) and PF (1, 10 and 100 μM). Subsequently, MTT assay was performed. The level of apoptosis was examined by flow cytometry and the oxidative stress was reflected by reactive oxygen species (ROS), malondialdehyde (MDA), glutathione S-transferases (GST) and glutathione peroxidase (GPX) levels. The mRNA expressions of Nuclear factor-E2-related factor 2 (Nrf2) and heme oxygenase 1 (HO-1) were detected by qRT-PCR. The levels of Kelch-like ECH-associating protein 1 (Keap1), Nrf2, HO-1, γ-glutamylcysteine synthetase (γGCS), B-cell CLL/lymphoma 2 (Bcl-2), Bax and Caspase 3 were detected by High content analysis and/or Western blot. Results The role of PF markedly suppressed high glucose induced Schwann cells oxidative stress by decreasing ROS and MDA levels and increasing GST and GPX activity. Western blot analysis showed that PF induced nuclear translocation of Nrf2. High content analysis showed that PF promoted Nrf2 dissociation from Keap1 and upregulating the Nrf2/ antioxidant response element (ARE) pathway. Furthermore, PF reduced Schwann cells apoptosis by increasing Bcl-2 and inhibiting Bax and Caspase-3 expressions. Conclusions PF in the management of Schwann cells oxidative stress induced by high glucose may be associated with activation of Nrf2/ARE pathway and Bcl-2-related apoptotic pathway. [ABSTRACT FROM AUTHOR]