P38 MAPK Upregulation Research Articles

Killer cell lectin-like receptor G2 facilitates aggressive phenotypes of gastric cancer cells via dual activation of the ERK1/2 and JAK/STAT pathways.

Advanced gastric cancer (GC) has a poor prognosis. This study aimed to identify novel GC-related genes as potential therapeutic targets. Killer cell lectin-like receptor G2 (KLRG2) was identified as a candidate gene by transcriptome analysis of metastatic GC tissues. Small interfering RNA-mediated KLRG2 knockdown in human GC cell lines was used to investigate KLRG2 involvement in signaling pathways and functional behaviors in vitro and in vivo. Clinicopathological data were analyzed in patients stratified according to tumor KLRG2 mRNA expression. KLRG2 knockdown in GC cells decreased cell proliferation, migration, and invasion; caused cell cycle arrest in G2/M phase; induced apoptosis via caspase activation; suppressed JAK/STAT and MAPK-ERK1/2 pathway activities; and upregulated p53 and p38 MAPK activities. In mouse xenograft models of peritoneal metastasis, the number and weight of disseminated GC nodules were decreased by KLRG2 knockdown. High tumor levels of KLRG2 mRNA were significantly associated with lower 5-year overall survival (OS) and relapse-free survival (RFS) rates in patients with Stage I-III GC (5-year OS rate: 64.4% vs. 80.0%, P = 0.009; 5-year RFS rate: 62.8% vs. 78.1%, P = 0.030). KLRG2 knockdown attenuated the malignant phenotypes of GC cells via downregulation of JAK/STAT and MAPK-ERK1/2 pathway activity and upregulation of p38 MAPK and p53. Targeted suppression of KLRG2 may serve as a new treatment approach for GC.

Interleukin-24-mediated antitumor effects against human glioblastoma via upregulation of P38 MAPK and endogenous TRAIL-induced apoptosis and LC3-II activation-dependent autophagy

BackgroundMelanoma differentiation-associated gene 7 (Mda-7) encodes IL-24, which can induce apoptosis in cancer cells. A novel gene therapy approach to treat deadly brain tumors, recombinant mda-7 adenovirus (Ad/mda-7) efficiently kills glioma cells. In this study, we investigated the factors affecting cell survival and apoptosis and autophagy mechanisms that destroy glioma cells by Ad/IL-24.MethodsHuman glioblastoma U87 cell line was exposed to a multiplicity of infections of Ad/IL-24. Antitumor activities of Ad/IL-24 were assessed by cell proliferation (MTT) and lactate dehydrogenase (LDH) release analysis. Using flow cytometry, cell cycle arrest and apoptosis were investigated. Using the ELISA method, the tumor necrosis factor (TNF-α) level was determined as an apoptosis-promoting factor and Survivin level as an anti-apoptotic factor. The expression levels of TNF-related apoptosis inducing ligand(TRAIL) and P38 MAPK genes were assessed by the Reverse transcription-quantitative polymerase chain reaction(RT‑qPCR) method. The expression levels of caspase-3 and protein light chain 3-II (LC3-II) proteins were analyzed by flow cytometry as intervening factors in the processes of apoptosis and autophagy in the cell death signaling pathway, respectively.ResultsThe present findings demonstrated that transduction of IL-24 inhibited cell proliferation and induced cell cycle arrest and cell apoptosis in glioblastoma. Compared with cells of the control groups, Ad/IL24-infected U87 cells exhibited significantly increased elevated caspase-3, and TNF-α levels, while the survivin expression was decreased. TRAIL was shown to be upregulated in tumor cells after Ad/IL-24 infection and studies of the apoptotic cascade regulators indicate that Ad/IL-24 could further enhance the activation of apoptosis through the TNF family of death receptors. In the current study, we demonstrate that P38 MAPK is significantly activated by IL-24 expression. In addition, the overexpression of mda-7/IL-24 in GBM cells induced autophagy, which was triggered by the upregulation of LC3-II.ConclusionsOur study demonstrates the antitumor effect of IL-24 on glioblastoma and may be a promising therapeutic approach for GBM cancer gene therapy.

Antiproliferative and apoptotic effect of tuna blood on human lung cancer A549 cells via p38 MAPKs and Akt pathway

Tuna blood has been reported the anti-inflammatory properties. However, the anticancer effects of tuna blood remain unelucidated. Therefore, this study investigated the anticancer effects of tuna blood against human non-small cell lung cancer (NSCLC) A549 cells and its potential mechanism of action. The amino acid profile presented in tuna whole blood freeze-dried (TWBF) and tuna blood cell freeze-dried (TRBF) were analyzed, and the antiproliferative effect was assessed by MTT assay. The nuclear staining, reactive oxygen species (ROS) generation, and mitochondrial membrane potential (MMP) were examined by Hoechst/PI staining, DCFHDA assay, and JC-1 staining. The expression of apoptosis-related proteins and the involvement of p38 MAPKs and Akt signaling were evaluated by western blotting. TWBF and TRBF inhibited the proliferation of lung cancer A549 cells with IC50 values of 354.97, 141.31 µg/mL, respectively. TRBF revealed more efficacy of cell proliferation inhibition than that of TWBF. The percentage of apoptotic cell death after treatment with 100 µg/mL TRBF was 35.95%. The increase in the ROS production and the disruption of MMP were also detected. At a mechanistic level, the exposure of TRBF to A549 cells showed an increase of the Bax/Bcl2 ratio and subsequently activated the proteolytic activity of caspase-3. TRBF administration induced apoptosis through the downregulation of protein kinase B (Akt) and upregulation of p38 MAPK. These findings indicate TRBF inhibits lung cancer cell proliferation and mediates apoptosis signaling, suggesting a promising chemotherapeutic candidate target NSCLC.

Janerin Induces Cell Cycle Arrest at the G2/M Phase and Promotes Apoptosis Involving the MAPK Pathway in THP-1, Leukemic Cell Line

Janerin is a cytotoxic sesquiterpene lactone that has been isolated and characterized from different species of the Centaurea genus. In this study, janerin was isolated form Centaurothamnus maximus, and its cytotoxic molecular mechanism was studied in THP-1 human leukemic cells. Janerin inhibited the proliferation of THP-1 cells in a dose-dependent manner. Janerin caused the cell cycle arrest at the G2/M phase by decreasing the CDK1/Cyclin-B complex. Subsequently, we found that janerin promoted THP-1 cell death through apoptosis as indicated by flow cytometry. Moreover, apoptosis induction was confirmed by the upregulation of Bax, cleaved PARP-1, and cleaved caspase 3 and the downregulation of an anti-apoptotic Bcl-2 biomarker. In addition, immunoblotting indicated a dose dependent upregulation of P38-MAPK and ERK1/2 phosphorylation during janerin treatment. In conclusion, we have demonstrated for the first time that janerin may be capable of inducing cell cycle arrest and apoptosis through the MAPK pathway, which would be one of the mechanisms underlying its anticancer activity. As a result, janerin has the potential to be used as a therapeutic agent for leukemia.

Astaxanthin alleviates inflammatory pain by regulating the p38 mitogen-activated protein kinase and nuclear factor-erythroid factor 2-related factor/heme oxygenase-1 pathways in mice

Inflammatory pain is a complex process that has a substantial negative impact on post-injury quality of life. Astaxanthin (AST), which is a lipid-soluble red-orange carotenoid that is found in lobsters, inhibits the development and maintenance of inflammation in mice via its antioxidant and anti-inflammatory activities. However, the specific mechanisms underlying these effects remain unclear. In this study, we aimed to elucidate the mechanism by which astaxanthin alleviated inflammation using a mouse model with Complete Freund's adjuvant (CFA)-induced inflammatory pain. Mechanical allodynia and thermal hyperalgesia were observed on days 1-14 post CFA injection. Expression of p38 mitogen-activated protein kinase (MAPK) in the left paw and L4-6 dorsal root ganglia (DRG) were upregulated in the CFA-induced mice. Expression of the nuclear factor erythroid 2-related factor 2 (Nrf2)/heme oxygenase-1 (HO-1) pathways were also increased. Astaxanthin relieved mechanical allodynia and thermal hyperalgesia induced by CFA and inhibited the inflammatory response (e.g., infiltration of inflammatory cells and production of inflammatory factors) in the ipsilateral paw and DRG. Additionally, AST inhibited p38 MAPK and enhanced Nrf2/HO-1 contents in the left paw and DRG, and reversed the pain induced by p38 MAPK agonist and Nrf2 inhibitors. These findings suggest that AST exerts anti-inflammatory effects and regulates p38 MAPK and Nrf2/HO-1 to alleviate inflammatory pain. AST may be a potential therapeutic agent for relieving inflammation.

The Impact of Royal Jelly against Hepatic Ischemia/Reperfusion-Induced Hepatocyte Damage in Rats: The Role of Cytoglobin, Nrf-2/HO-1/COX-4, and P38-MAPK/NF-κB-p65/TNF-α Signaling Pathways.

The present study was conducted to elucidate the underlying molecular mechanism as well as the potential hepatoprotective effects of royal jelly (RJ) against hepatic ischemia/ reperfusion (IR) injury. Rats were assigned into four groups; sham (received vehicle), IR (30 minutes ischemia and 45 minutes reperfusion), sham pretreated with RJ (200 mg/kg P.O.), and IR pretreated with RJ (200 mg/kg P.O.). The experiment lasted for 28 days. Hepatic IR significantly induced hepatic dysfunctions, as manifested by elevation of serum transaminases, ALP and LDH levels. Moreover, hepatic IR caused a significant up-regulation of P38-MAPK, NF-κB-p65, TNF-α and MDA levels along with marked down-regulation of Nrf-2, HO-1, COX-4, cytoglobin, IκBa, IL-10, GSH, GST and SOD levels. Additionally, marked histopathological changes were observed after hepatic IR injury. On the contrary, pretreatment with RJ significantly improved hepatic functions along with the alleviation of histopathological changes. Moreover, RJ restored oxidant/antioxidant balance as well as hepatic expressions of Nrf- 2, HO-1, COX-4, and cytoglobin. Simultaneously, RJ significantly mitigated the inflammatory response by down-regulation of P38-MAPK, NF-κB-p65, TNF-α expression. The present results revealed that RJ has successfully protected the liver against hepatic IR injury through modulation of cytoglobin, Nrf-2/HO-1/COX-4, and P38-MAPK/NF-κB-p65/TNF- α signaling pathways.

Shikimic acid, a mannose bioisostere, promotes hair growth with the induction of anagen hair cycle

Shikimic acid (SA) has recently been found to be a major component of plant stem cells. The exact effects of SA on human hair follicles (HFs) is unknown. The purpose of this study was to examine the effects of SA on hair growth. We investigated the effect of SA on an in vivo C57BL/6 mouse model. We examined the expression of mannose receptor (MR), which is a known receptor of SA, in human HFs and the effect of SA on human dermal papilla cells (hDPCs), outer root sheath cells (hORSCs), and on ex vivo human hair organ culture. SA significantly prolonged anagen hair growth in the in vivo mouse model. We confirmed expression of the MR in human HFs, and that SA increased the proliferation of hDPCs and hORSCs. It was found that SA enhanced hair shaft elongation in an ex vivo human hair organ culture. SA treatment of hDPCs led to increased c-myc, hepatocyte growth factor, keratinocyte growth factor and vascular endothelial growth factor levels and upregulation of p38 MAPK and cAMP response element-binding protein levels. Our results show that SA promotes hair growth and may serve as a new therapeutic agent in the treatment of alopecia.

Delving into the Antiurolithiatic Potential of Tribulus terrestris Extract Through \u2013In Vivo Efficacy and Preclinical Safety Investigations in Wistar Rats

Modern treatment interventions for kidney stones are wrought with side-effects, hence the need for alternative therapies such as plant-based medicines. We have previously documented through in vitro studies that statistically optimized aqueous extract of Tribulus terrestris (Zygophyllaceae family) possesses antiurolithic and antioxidant potential. This provides strong scientific foundation to conduct in vivo efficacy and preclinical safety studies to corroborate and lend further proof to its ability to prevent and cure kidney stones. The preventive and curative urolithiatic efficacy in experimentally induced nephrolithiatic Wistar rats, along with preclinical toxicity was evaluated following oral administration of statistically optimized aqueous extract of T. terrestris. Treatment showed augmented renal function, restoration of normal renal architecture and increase in body weight. Microscopic analysis of urine revealed excretion of small sized urinary crystals, demonstrating that treatment potentially modulated the morphology of renal stones. Tissue enzymatic estimation affirmed the antioxidant efficacy of treatment with reduced free radical generation. Significant upregulation of p38MAPK at both the gene and protein level was noted in hyperoxaluric group and interestingly treatment reversed it. Acute oral toxicity study established the Median Lethal Dose (LD50) to be greater than 2000 mg/kg body weight (b.wt.) No observed adverse effect level (NOAEL) by repeated oral toxicity for 28 days at 750 mg/kg b.wt. was noted. This study lends scientific evidence to the safe, preventive and curative potential of statistically optimized aqueous extract of T. terrestris at a dose of 750 mg/kg b.wt. and suggests that the extract shows promise as a therapeutic antiurolithic agent.

PKR Promotes Oxidative Stress and Apoptosis of Human Articular Chondrocytes by Causing Mitochondrial Dysfunction through p38 MAPK Activation-PKR Activation Causes Apoptosis in Human Chondrocytes.

Osteoarthritis (OA) is one of the most common types of arthritis in the elderly people. It has been known that chondrocyte apoptosis occurs in OA cartilage; however, the detailed molecular mechanism remains unclear. In the current study, we aimed to elucidate the role of double-stranded RNA-dependent protein kinase R (PKR) in the TNF-α-caused apoptosis in chondrocytes. Human articular chondrocytes were digested from cartilages of OA subjects who accepted arthroplastic knee surgery. Our results showed that phosphorylation of p38 MAPK was increased after TNF-α stimulation or PKR activation using poly (I:C), and TNF-α-induced p38 MAPK upregulation was inhibited by PKR inhibition, suggesting phosphor-p38 MAPK was regulated by PKR. Moreover, we found that PKR participated in the p53-dependent destruction of AKT following activation of p38 MAPK. The inhibition of AKT led to the reduced expression of PGC-1α, which resulted in mitochondrial dysfunction and increased oxidative stress. We showed that the reduction of oxidative stress using antioxidant Mito TEMPO lowered the TNF-α-induced caspase-3 activation and TUNEL-positive apoptotic cells. The diminished apoptotic response was also observed after repression of PKR/p38 MAPK/p53/AKT/PGC-1α signaling. Taken together, we demonstrated that the aberrant mitochondrial biogenesis and increased oxidative stress in chondrocytes after TNF-α stimulation were mediated by PKR, which may contribute to the chondrocyte apoptosis and cartilage degeneration in OA.

English

Several scientific reports documented the potent anti-cancer effect of various natural products  such as curcumin, epigallocatechin gallate (EGCG), resveretrol via the rise in reactive oxygen species (ROS) and dysfunction of anti-apoptotic gene expressions. The present study evaluated the anticancer potential of lotus derived alkaloids, neferine and isoliensinine on colorectal cancer HCT-15 (CRCs) cells. Neferine/Isoliensinine treatments induced cytotoxicity with sequential enhancement of intracellular ROS, intracellular calcium [Ca2+]i  and mitochondrial membrane potential (ΔψM). Furthermore, cell cycle, Annexin FITC and PI uptake were carried out by flow cytometry. Activation of p38 MAPK and apoptotic genes expression was done by western blotting and reverse transcription polymerase chain reaction (RT-PCR) respectively. Results indicate neferine/isoliensinine induced hyper-generation of ROS was responsible for their cytotoxic effect in CRCs consequently, with a significant increase in [Ca2+]i followed by a significant decrease in the ΔψM. The above treatments induced cell cycle arrest at G1 phase whereas apoptosis was indicated by upregulation of p38 MAPK, Bax, caspase 9, caspase 3, cleaved poly (ADP-ribose) polymerase (PARP) and down-regulation of Bcl2. In conclusion, this is the first study of anticancer effect of neferine/isoliensinine via apoptotic mechanism in CRCs and the results suggest that neferine/isoliensinine induced apoptosis through the ROS generation, activation of p38 MAPK which in turn induces mitochondrial mediated apoptosis. Key words: Neferine, isoliensinine, reactive oxygen species (ROS), HCT-15, apoptosis. &nbsp

The role of adenosine in up-regulation of p38 MAPK and ERK during limb ischemic preconditioning-induced brain ischemic tolerance

Our previous studies have demonstrated that limb ischemic preconditioning (LIP) induced brain ischemic tolerance and up-regulated the expression of p38 MAPK and ERK in the hippocampal CA1 region in rats. The present study was undertaken to investigate the role of adenosine in brain protection and up-regulation of p38 MAPK and ERK induced by LIP. It was found that adenosine A1 receptor antagonist DPCPX dose-dependently inhibited the protective effect of LIP. The up-regulation of p38 MAPK and ERK induced by LIP could be blocked by DPCPX. Furthermore, we observed the effect of adenosine on the brain ischemia. The results showed that pre-administration of adenosine could partly mimic the neuroprotective effect on the brain, up-regulate the expression of p38 MAPK and ERK. Based on the above results, it can be concluded that adenosine participated in brain protection and up-regulation of the expression of p38 MAPK and ERK during the induction of brain ischemic tolerance after LIP.

The role of chondroitin sulfate in regulating hypertrophy during MSC chondrogenesis in a cartilage mimetic hydrogel under dynamic loading

Mesenchymal stem cells (MSCs) are promising for cartilage regeneration, but readily undergo terminal differentiation. The aim of this study was two-fold: a) investigate physiochemical cues from a cartilage-mimetic hydrogel under dynamic compressive loading on MSC chondrogenesis and hypertrophy and b) identify whether Smad signaling and p38 MAPK signaling mediate hypertrophy during MSC chondrogenesis. Human MSCs were encapsulated in photoclickable poly(ethylene glycol) hydrogels containing chondroitin sulfate and RGD, cultured under dynamic compressive loading or free swelling for three weeks, and evaluated by qPCR and immunohistochemistry. Loading inhibited hypertrophy in the cartilage-mimetic hydrogel indicated by a reduction in pSmad 1/5/8, Runx2, and collagen X proteins, while maintaining chondrogenesis by pSmad 2/3 and collagen II proteins. Inhibiting pSmad 1/5/8 under free swelling culture significantly reduced collagen X protein, similar to the loading condition. Chondroitin sulfate was necessary for load-inhibited hypertrophy and correlated with enhanced S100A4 expression, which is downstream of the osmotic responsive transcription factor NFAT5. Inhibiting p38 MAPK under loading reduced S100A4 expression, and upregulated Runx2 and collagen X protein. Findings from this study indicate that chondroitin sulfate with dynamic loading create physiochemical cues that support MSC chondrogenesis and attenuate hypertrophy through Smad 1/5/8 inhibition and p38 MAPK upregulation.

The effect of "NutramilTM Complex," food for special medical purpose, on breast and prostate carcinoma cells.

NutramilTM Complex is a multicomponent food product that meets the requirements of a food for special medical purpose. As a complete, high-energy diet it consists of properly balanced nutrients, vitamins and minerals. The aim of this study was to assess the effect of NutramilTM Complex on breast and prostate carcinoma cells. Our results showed that NutramilTM Complex reduced the viability and proliferation of breast and prostate cancer cells and that this process was associated with the induction of apoptosis via activation of caspase signalling. Data showed elevated levels of p53 tumour suppressor, up-regulation of p38 MAPK and SAPK / JNK proteins and downregulation of anti-apoptotic ERK1/2, AKT1 and HSP27. Treatment with NutramilTM Complex also affected the expression of the BCL2 family genes. Results also showed down-regulation of anti-apoptotic BCL-2 and up-regulation of pro-apoptotic members such as BAX, BAD, BID. In addition, we also observed regulation of many other genes, including Iκβα, Chk1 and Chk2, associated with apoptotic events. Taken together, our results suggest activation of the mitochondrial apoptotic pathway as most likely mechanism of anti-carcinogenic activity of NutramilTM Complex.

The experimental research on anti-glioma with temozolomide, all-trans retinoic acid and radiotherapy

Objective To investigate whether the collaborative application of temozolomide (TMZ), all-trans retinoic acid (ATRA) and radiotherapy can induce the apoptosis of U251 glioma cells and the molecular mechanism. Methods We detected the cell apoptosis rate of control group (100 μl nutrient solution), radiotherapy group (radiation dosage of 8 Gy), medicine group (TMZ 200 μmol/L+ ATRA 20 μmol/L) and combined group (radiation dosage of 8 Gy+ TMZ 200 μmol/L+ ATRA 20 μmol/L) by flow cytometry method. The protein expression of p38 mitogen-activated protein kinase (MAPK) and nuclear factor-κB (NF-κB) was detected by Western blotting. Results The apoptosis rate was (9.96±3.78)%, (37.35±5.36)%, (59.89±7.15)% and (69.35±8.25)% in control group, radiotherapy group, medicine group and combined group respectively. The apoptosis rate in combined group was higher than that in other groupsThese differences were more obvious (χ2=7 471.175, P=0.000; χ2=2 057.235, P=0.000; χ2=195.717, P=0.000). Combined group can up-regulate the protein expression of p38MAPK in contrast to control guoup, radiation group and medicine group. These differences were more obvious (t=46.000, P=0.000; t=10.150, P=0.000; t=4.266, P=0.013). Combined group can down-regulate the protein expression of NF-κB in contrast to control guoup, radiation group and medicine group. These differences were more obvious (t=36.380, P=0.000; t=30.090, P=0.000; t=68.620, P=0.000). Conclusion The combined use of TMZ, ATRA and radiotherapy can induce the apoptosis of U251 glioma cells probably by the upregulation of p38MAPK and downregulation of NF-κB. Key words: Glioma; Apoptosis; Temozolomide; All-trans retinoic acid; Radiotherapy; p38 mitogen-activated protein kinase; Nuclear factor-κB

Pro-inflammatory Signaling in a 3D Organotypic Skin Model after Low LET Irradiation-NF-κB, COX-2 Activation, and Impact on Cell Differentiation.

Nearly 85% of radiotherapy patients develop acute radiation dermatitis, which is an inflammatory reaction of the skin at the treatment field and in the surrounding area. The aims of this study were to unravel the mechanisms of radiation-induced inflammatory responses after localized irradiation in a human 3D organotypic skin culture model. This could provide possible inflammatory targets for reduction of skin side effects. 3D organotypic skin cultures were set up and locally irradiated with 225 kVp X-rays, using a combination of full exposure and partial shielding (50%) of the cultures. The secretion of pro-inflammatory cytokines, the phenotype, and the differentiation markers expression of the cultures were assessed up to 10 days postirradiation. The pro-inflammatory transcription factor nuclear factor kappa B (NF-κB) and cyclooxygenase-2 (COX-2) pathways have been studied. The results showed fast activation of NF-κB, most likely triggered by DNA damage in the irradiated cells, followed by upregulation of p38 MAPK and COX-2 in the irradiated and surrounding, non-irradiated, areas of the 3D cultures. The application of the COX-2 inhibitor sc-236 was effective at reducing the COX-2 mRNA levels 4 h postirradiation. The same inhibitor also suppressed the PGE2 secretion significantly 72 h after the treatment. The expression of a pro-inflammatory phenotype and abnormal differentiation markers of the cultures were also reduced. However, the use of an NF-κB inhibitor (Bay 11-7085) did not have the predicted positive effect on the cultures phenotype postirradiation. Radiation-induced pro-inflammatory responses have been observed in the 3D skin model. The activated signaling pathways involved NF-κB transcription factor and its downstream target COX-2. Further experiments aiming to suppress the inflammatory response via specific inhibitors showed that COX-2 is a suitable target for reduction of the normal skin inflammatory responses at radiotherapy, while NF-κB inhibition had detrimental effects on the 3D skin model development.

Expression of p38 mitogen-activated protein kinase (p38MAPK) and pathological change in intussusception.

The aim of this study was to develop a mouse model and further assess the pathological changes associated with the expression of p38mitogen-activated protein kinase (p38MAPK) in intussusception. Sixty-two adult Balb/C mice were used. A longitudinal incision was made in the middle rectus muscle in the body cavity. The ileum was intussuscepted into the colon. Measurements were taken at the onset of intussusception and at 5, 15, 30, 60, and 120 min. Mucosal impairment was assessed on microscopy. Ten of the intussuscepted mice were used as an ischemia-reperfusion (I/R) model. Immunohistochemistry was used to assess expression of p38MAPK in the I/R model and pediatric patients specimens of intussusception. The intussusception model was successfully established in 46 mice. After 15 min, vascular compromise became visible in these 46 mice. Over time, vascular function worsened. There were significant differences in microscopy injury score in the intestinal mucosa between the 15 min and 30 min groups (P = 0.0006), 30 min and 60 min groups (P = 0.0046), and the 60 min and 120 min groups (P = 0.0050). There was no significant difference between the 5 min and 15 min groups (P = 0.0597). p38MAPK was expressed strongly in pediatric specimens of intussusception. Immunostained sections of intestinal epithelium had significantly higher mean quick score for p38MAPK in the intussusception I/R model group than in the intussusception group and controls (P = 0.0130). On each two-group comparison there was a significant difference between groups (all P < 0.01; Fig.). The present mouse model can be used to assess the dynamic pathological changes associated with intussusception. I/R is associated with upregulation of p38MAPK in intussusception.

SVCT2, a potential therapeutic target, protects against oxidative stress during ethanol-induced neurotoxicity via JNK/p38 MAPKs, NF-κB and miRNA125a-5p

Sodium vitamin C transporter 2 (SVCT2) plays a key role in transporting ascorbic acid (AA), an important intracellular antioxidant, into neurons. It is well known that ethanol (EtOH) abuse causes significant neurodegeneration, as well as endogenous AA release in certain encephalic regions. Here, we identified that SVCT2 forms part of a self-defense mechanism that protects against oxidative stress in binge drinking rats, and SVCT2 levels are correlated with antioxidants and neuronal injury. Four days of binge drinking led to massive neuron degeneration in prefrontal cortex (PFC), accompanied by increased levels of 4-hydroxynonenal (4-HNE)-adducted proteins and SVCT2 expression, as well as dramatic changes in AA levels in rat brain. AA levels were decreased in PFC and increased in cerebrospinal fluid (CSF) after binge drinking, but returned to normal on the 7th day following EtOH withdrawal. These processes were further evaluated in primary cortical neurons exposed to 100mM EtOH in vitro. Neurons transfected with SVCT2 siRNA were more susceptible than controls to certain aspects of EtOH-induced injury, including cell death, dendrite damage and increased oxidative stress. EtOH-induced up-regulation of SVCT2 was associated with activation of JNK and p38 MAPKs and the NF-κB pathway. More importantly, miRNA-125a-5p was down-regulated in PFC of 4-day binge drinking rats and negatively regulated protein expression during EtOH-induced neuronal injury. MiR-125a-5p over-expression attenuated intracellular AA levels, promoted cell death and suppressed the EtOH-induced up-regulation of p38 MAPK and SVCT2, which suggested that miR-125a-5p plays an important role in SVCT2 function in EtOH-induced neuronal injury. We speculate that SVCT2, possibly regulated by JNK/p38 MAPKs, NF-κB signaling and miR-125a-5p, has a neuroprotective effect against EtOH-induced oxidative stress. Promotion of SVCT2 expression or stimulation of SVCT2 activity may be a promising therapeutic strategy for the prevention of EtOH-associated neurodegeneration.

Berberine induces neuronal differentiation through inhibition of cancer stemness and epithelial-mesenchymal transition in neuroblastoma cells

BackgroundBerberine, a plant alkaloid, has been used since many years for treatment of gastrointestinal disorders. It also shows promising medicinal use against metabolic disorders, neurodegenerative disorders and cancer; however its efficacy in neuroblastoma (NB) is poorly explored. HypothesisEMT is important in cancer stemness and metastasis resulting in failure to differentiate; thus targeting EMT and related pathways can have clinical benefits. Study designPotential of berberine was investigated for (i) neuronal differentiation and cancer stemness inhibition, (ii) underlying molecular mechanisms regulating cancer-stemness and (iii) EMT reversal. MethodsUsing neuro2a (N2a) neuroblastoma cells (NB); we investigated effect of berberine on neuronal differentiation, cancer-stemness, EMT and underlying signalling by immunofluorescence, RT-PCR, Western blot. High glucose-induced TGF-β mediated EMT model was used to test EMT reversal potential by Western blot and RT-PCR. STRING analysis was done to determine and validate functional protein-interaction networks. ResultsWe demonstrate berberine induces neuronal differentiation accompanying increased neuronal differentiation markers like MAP2, β-III tubulin and NCAM; generated neurons were viable. Berberine attenuated cancer stemness markers CD133, β-catenin, n-myc, sox2, notch2 and nestin. Berberine potentiated G0/G1 cell cycle arrest by inhibiting proliferation, cyclin dependent kinases and cyclins resulting in apoptosis through increased bax/bcl-2 ratio. Restoration of tumor suppressor proteins, p27 and p53, indicate promising anti-cancer property. The induction of NCAM and reduction in its polysialylation indicates anti-migratory potential which is supported by down regulation of MMP-2/9. It increased epithelial marker laminin and smad and increased Hsp70 levels also suggest its protective role. Molecular insights revealed that berberine regulates EMT via downregulation of PI3/Akt and Ras-Raf-ERK signalling and subsequent upregulation of p38-MAPK. TGF-β secretion from N2a cells was potentiated by high glucose and negatively regulated by berberine through modulation of TGF-β receptors II and III. Berberine reverted mesenchymal markers, vimentin and fibronectin, with restoration of epithelial marker E-cadherin, highlighting the role of berberine in reversal of EMT. ConclusionCollectively, the study demonstrates prospective use of berberine against neuroblastoma as elucidated through inhibition of fundamental characteristics of cancer stem cells: tumorigenicity and failure to differentiation and instigates reversal in the EMT.

DEPTOR promotes survival of cervical squamous cell carcinoma cells and its silencing induces apoptosis through downregulating PI3K/AKT and by up-regulating p38 MAP kinase.

DEPTOR is an endogenous inhibitor of mTOR complexes, de-regulated in cancers. The present study reveals a vital role for DEPTOR in survival of cervical squamous cell carcinoma (SCC). DEPTOR was found to be overexpressed in both cervical SCC cells and tissues and it's silencing in cervical SCC cells induced apoptosis, mainly by up-regulation of p38 MAPK and by inhibiting PI3K/AKT pathway via a feed-back inhibition from mTORC1-S6K. DEPTOR silencing resulted in reduced expression of the nitric oxide synthases iNOS and eNOS, as well as increased activation of ERK1/2 and p38 MAP kinases. Activation of AKT signaling by overexpression of constitutively active-AKT (CA-AKT) failed to overcome the apoptosis caused by DEPTOR silencing. Similarly pharmacological inhibition of ERK also failed to control apoptosis. However pharmacological inhibition of p38 MAPK rescued the cells from apoptosis, indicating the major role of p38 MAPK in cell death induced by DEPTOR silencing. DEPTOR was also found to regulate ERK1/2 in an AKT dependent manner. DEPTOR knockdown induced cell death in SiHa cells overexpressing the anti-apoptotic Bcl-2 and Bcl-xL, indicating strong survival role of DEPTOR in these cells. DEPTOR overexpression activated PI3K/AKT by relieving the negative feed-back inhibition from mTORC1-S6K. DEPTOR regulation was also observed to be independent of HPV E6/E7 oncoproteins, but it might be a molecular co-factor contributing to cervical carcinogenesis. In summary, DEPTOR is found to promote survival of cervical SCC cells and its reduction induced apoptosis via differential effects on PI3K/AKT and p38 MAPK and can be a potential target in cervical SCC.

Loss of DGKε induces endothelial cell activation and death independently of complement activation

AbstractAtypical hemolytic uremic syndrome (aHUS) is classically described to result from a dysregulation of the complement alternative pathway, leading to glomerular endothelial cell (EC) damage and thrombosis. However, recent findings in families with aHUS of mutations in the DGKE gene, which is not an integral component of the complement cascade, led us to consider other pathophysiologic mechanisms for this disease. Here, we demonstrate that loss of DGKε expression/activity in EC induces an increase in ICAM-1 and tissue factor expression through the upregulation of p38-MAPK–mediated signals, thus highlighting a proinflammatory and prothrombotic phenotype of DGKε-deficient ECs. More interestingly, DGKE silencing also increases EC apoptosis and impairs EC migration and angiogenesis in vitro, suggesting that DGKE loss-of-function mutations impair EC repair and angiogenesis in vivo. Conversely, DGKE knockdown moderately decreases the expression of the complement inhibitory protein MCP on quiescent EC, but does not induce complement deposition on their surface in vitro. Collectively, our data strongly suggest that in DGKE-associated aHUS patients, thrombotic microangiopathy results from impaired EC proliferation and angiogenesis rather than complement-mediated EC lesions. Our study expands the current knowledge of aHUS mechanisms and has implications for the treatment of patients with isolated DGKE mutations.