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Background: Oxaliplatin resistance is a challenge in treating colorectal cancer (CRC) patients, contributory to the failure in chemotherapy. However, the mechanism of Oxaliplatin resistance has not been completely elucidated. In this study, we explored the key molecule involved in the Oxaliplatin resistance, which could be a candidate therapeutic target to attenuate chemo-resistance in CRC cells.Methods: Microarray screening, western blot and qPCR on clinic CRC samples were conducted to select the target gene ABCC10. The Oncomine Oncology Database and the Cancer Genome Atlas (TCGA) data were analyzed to figure out the correlation between the clinical manifestation and ABCC10. ABCC10 knock-down in CRC cells was conducted to identify its role in the Oxaliplatin resistance. Cell counting kit-8 (CCK-8) assay was conducted to identify the CRC cell viability and Oxaliplatin IC50. Flow cytometry was conducted to detect the cell apoptosis exposed to Oxaliplatin. The intracellular Oxaliplatin accumulation was measured by ultra-high performance liquid chromatography coupled to tandem mass spectrometry (UPLC-MS/MS).Results: ABCC10 transporter was correlated with CRC relapse and metastasis, indicating a CRC-promoting effect of ABCC10. In ABCC10 knock-down CRC cells the Oxaliplatin sensitivity was significantly elevated due to an increase of intracellular Oxaliplatin accumulation resulted from the diminished drug efflux. We next explored a strategy to inhibit ABCC10 in CRC cells, paying a special interest in the endoplasmic reticulum stress (ERS) / unfolded protein response (UPR) that plays a dual role in tumor development. We found that the CRC cell viability was profoundly decreased and the pro-apoptotic factor CHOP and apoptosis were increased by the intense ERS/UPR instead of the inhibited and mild ERS/UPR. Significantly, the Oxaliplatin sensitivity of CRC cells was enhanced in response to the intense ERS, which was blocked by inhibiting IRE1α branch of UPR. Finally, we figured out that the intense ERS/UPR down-regulated ABCC10 via regulated IRE1-dependent decay (RIDD) activity. Conclusion: Oxaliplatin was a substrate of ABCC10. The intense ERS/IRE1α elicited anti-CRC effects through down-regulating ABCC10 so as to increase Oxaliplatin sensitivity, in addition to inducing CHOP. We suggested that introduction of intense ERS/UPR could be a promising strategy to restore chemo-sensitivity when used in combination with Oxaliplatin or other chemotherapeutic drugs pumped out by ABCC10.

Zika virus (ZIKV) belongs to mosquito-borne flaviviruses. Unlike other members in the family, ZIKV can be sexually transmitted, and the female genital tracts are susceptible to ZIKV. However, the impacts of ZIKV infection on nonpregnant female reproductive health are not understood. In this study, we investigated the effects of ZIKV infection on the ovary by using nonpregnant female interferon α/β receptor-deficient (Ifnar1-/-) mice. The results showed that the ovary supported ZIKV replication, and the granulosa and theca cells of antral follicles were susceptible. ZIKV replication in situ significantly reduced the numbers of antral follicles, aggravated follicular atresia and disrupted folliculogenesis. Notably, ZIKV replication in the ovary caused disordered ovarian steroidogenesis manifested by decreased expression of key enzymes linked to sex hormone synthesis including the cytochrome P450 17A1 (CYP17A1) and aromatase (CYP19A1). Further, we observed that ZIKV infection disrupted the estrous cycle, and thus prolonged the time to conceive. More importantly, although ZIKV RNA could not be detected at 3 months post infection, the damaged ovarian structure and dysfunction were also observed. Taken together, our study demonstrates that ZIKV infection in nonpregnant female mice cause ovarian damage and dysfunction, even long after ZIKV clearance. These data provide important information to understand the effects of ZIKV infection in female reproductive tissues and basic evidence for further studies. IMPORTANCE ZIKV, a flavivirus, is primarily transmitted by mosquito bites. But it can also be transmitted vertically and sexually. Although ZIKV-associated Guillain-Barre syndrome and microcephaly have drawn great attention, there have been few studies on the potential effects of ZIKV on genital tract of non-pregnant female. This study investigated the effects of ZIKV on the ovary in mice. We found that ZIKV replicated in the ovary and the granulosa and theca cells of antral follicles were susceptible. ZIKV replication in situ significantly damaged ovarian structure and function, and disrupted folliculogenesis. Notably, ZIKV infection further disrupted the estrous cycle and prolonged the time to conceive in mice by causing disordered ovarian steroidogenesis. These effects were observed in both the acute phase and the recovery phase after viral elimination. Overall, the new findings provide important additions to make out the potential adverse impacts of ZIKV on reproductive health in females.

The risk of colitis and colorectal cancer increases markedly throughout adult life, endangering the health and lives of elderly individuals. Previous studies have proposed that bacterial translocation and infection are the main risk factors for these diseases. Therefore, in the present study, we aimed to identify the underlying mechanism by focusing on the mucus barrier function and mucin‐type O‐glycosylation. We evaluated alterations in the colon mucus layer in 2‐, 16‐, and 24‐month‐old mice and aged humans. Aged colons showed defective intestinal mucosal barrier and changed mucus properties. The miR‐124‐3p expression level was significantly increased in the aged distal colonic mucosa, which was accompanied by an increase in pathogens and bacterial translocation. Meanwhile, T‐synthase, the rate‐limiting enzyme in O‐glycosylation, displayed an age‐related decline in protein expression. Further experiments indicated that miR‐124‐3p modulated O‐glycosylation by directly targeting T‐synthase. Moreover, young mice overexpressing miR‐124‐3p exhibited abnormal glycosylation, early‐onset, and more severe colitis. These data suggest that miR‐124‐3p predisposes to senile colitis by reducing T‐synthase, and the miR‐124‐3p/T‐synthase/O‐glycans axis plays an essential role in maintaining the physiochemical properties of colonic mucus and intestinal homeostasis., Young colons show intact inner layer and mucin‐type O‐glycosylation, limiting microbiota access to the epithelium. Age‐related alteration in miR‐124‐3p/T‐synthase/O‐glycans axis and reduction in mucus thickness weakened the mucus barrier, leading to abnormal bacterial intrusion and increased susceptibility to senile colitis.

Low expression of the tumor suppressor Kelch-like ECH-associated protein 1 (KEAP1) in non–small cell lung cancer (NSCLC) often results in higher malignant biological behavior and poor prognosis; however, the underlying mechanism remains unclear. The present study demonstrates that overexpression of Keap1 significantly suppresses migration and invasion of three different lung cancer cells (A549, H460, and H1299). Highly expressed Keap1, compared with the control, promotes formation of multiple stress fibers with larger mature focal adhesion complexes in the cytoplasm where only fine focal adhesions were observed in the membrane under control conditions. RhoA activity significantly increased when Keap1 was overexpressed, whereas Myosin 9b expression was reduced but could be rescued by proteasome inhibition. Noticeably, mouse tumor xenografts with Keap1 overexpression were smaller in size and less metastatic relative to the control group. Taken together, these results demonstrate that Keap1 stabilizes F-actin cytoskeleton structures and inhibits focal adhesion turnover, thereby restraining the migration and invasion of NSCLC. Therefore, increasing Keap1 or targeting its downstream molecules might provide potential therapeutic benefits for the treatment of patients with NSCLC. Implications: This study provides mechanistic insight on the metastatic process in NSCLC and suggests that Keap1 and its downstream molecules may be valuable drug targets for NSCLC patients. Mol Cancer Res; 16(3); 508–16. ©2018 AACR.

Macrophage receptor with collagenous structure (MARCO) is a member of the class A scavenger receptor family which is expressed on the cell surface of macrophages. It is well known that MARCO avidly binds to unopsonized pathogens, leading to its ingestion by macrophages. However, the role of MARCO in the recognition and phagocytosis of tumor cells by macrophages remains poorly understood. In this study, we used lentiviral technology to knockdown and overexpress MARCO and investigated the ability of macrophages to phagocytose tumor cells. Our results showed that MARCO expression was correlated with the ability of macrophages to carry on phagocytosis. MARCO knockdown led to significant decreases in the number of engulfment pseudopodia of macrophages and inhibition of the phagocytosis of tumor cells. On the other hand, MARCO overexpression elevated activity of SYK, PI3K and Rac1 in macrophages, which led to changes in macrophage morphology and enhanced phagocytosis by promoting formation of stress fibers and pseudopodia. By Co-IP analysis we showed that MARCO directly binds to the β5 integrin of SL4 tumor cells. In summary, these results demonstrated the important role for MARCO in demonstrated tumor cells uptake and clearance by macrophages.

It was reported that Src-mediated and RTK-dependent accumulation of key transcription factor, ETV4, which played an important role in the migration of embryonic cells and tumor cells, were regulated by their common downstream MAPK molecules. However, the detailed mechanism was not completely clear. In the present study, we revealed that ETV4 protein was significantly enhanced by ERK kinase activation in the colorectal cancer (CRC) patients and mouse models as well as in the CRC cell lines. It was further confirmed that the activation of ERK kinase led to the phosphorylation of ETV4 at Ser73 and the ETV4 phosphorylation could block its binding to COP1, thereby stabilized ETV4 via avoiding its ubiquitination degradation. In addition, this effect was not due to altering an E3 ubiquitin ligase, COP1 amount or p-COP1/COP1 ratio. Our results will help understand the mechanism of ETV4 overexpression in CRC patients and provide a clue to search new therapeutic target to treat the related tumors in clinical practice.

Acupuncture is a therapy effective in treating postoperative ileus (POI); its underlying mechanisms remain unclear. MicroRNAs (miRNAs) participate in inflammation and injury to the interstitial cells of Cajal (ICCs), both of which are considered to be contributors to POI. C-kit, encoding KIT, a specific marker of ICCs, is predicted to be targeted by miR-19a, an inflammation-related miRNA. Therefore, we investigated a possible link between inflammation, miR-19a, and ICCs in POI, as well as the mechanism by which these factors are affected by acupuncture. The effects of acupuncture on POI were assessed in patients after colorectal resection and in colocolic anastomosis mice. Immunofluorescence staining demonstrated that KIT[Formula: see text]/ano1[Formula: see text] ICCs dramatically decreased around the colonic incision in mice, which was negatively correlated with the pronounced increase in macrophage. However, this decrease was not due to apoptosis. IL-6R was expressed in ICCs, and IL-6 level was significantly increased, as measured by ELISA, in accompaniment with high miR-19a expression. The increase in IL-6 and miR-19a levels was negatively correlated with the decrease in KIT[Formula: see text]/ano1[Formula: see text] ICCs. A luciferase reporter assay demonstrated that miR-19a directly targeted C-kit, indicating that miR-19a caused ICC damage. Interestingly, acupuncture inhibited macrophage activation, IL-6 release, and miR-19a upregulation, while promoting KIT and ano1 restoration in ICCs. High serum miR-19a level in patients after colorectal resection was also reduced by acupuncture. Conclusively, the IL-6 released by macrophages during gastrointestinal surgery upregulated miR-19a, which downregulated KIT in ICCs and finally resulted in POI. Acupuncture can interfere with the “IL-6–miR-19a–KIT” axis, suggesting that it may be a therapeutic mechanism that works against POI.

Gastrointestinal (GI) distention is a common pathological characteristic in most GI motility disorders (GMDs), however, their detail mechanism remains unknown. In this study, we focused on Ca2+ overload of smooth muscle, which is an early intracellular reaction to stretch, and its downstream MAPK signaling and also reduction of SCF in vivo and in vitro. We successfully established colonic dilation mouse model by keeping incomplete colon obstruction for 8 days. The results showed that persistent colonic dilation clearly induced Ca2+ overload and activated all the three MAPK family members including JNK, ERK and p38 in smooth muscle tissues. Similar results were obtained from dilated colon of patients with Hirschsprung's disease and stretched primary mouse colonic smooth muscle cells (SMCs). Furthermore, we demonstrated that persistent stretch-induced Ca2+ overload was originated from extracellular Ca2+ influx and endoplasmic reticulum (ER) Ca2+ release identified by treating with different Ca2+ channel blockers, and was responsible for the persistent activation of MAPK signaling and SCF reduction in colonic SMCs. Our results suggested that Ca2+ overload caused by smooth muscle stretch led to persistent activation of MAPK signaling which might contribute to the decrease of SCF and development of the GMDs.

Cervical cancer is one of the most lethal types of cancer in female. Aberrant activation of Wnt/β-catenin signaling pathway has been found to be involved in cervical cancer development and progression, whereas the underlying molecular mechanisms remain poorly understood. The present study showed that NHERF1 was a novel gene associated with both cell proliferation and Wnt signaling pathway in cervical cancer by analysis of differential gene expression and gene cluster for the cervical cancer specimens from GEO data sets. It was further demonstrated in cellular study that NHERF1 inhibition of cervical cancer cell proliferation through Wnt/β-catenin signaling was dependent on α-actinin-4 (ACTN4) expression. A negative association between NHERF1 expression and levels of ACTN4 and β-catenin was found in mouse xenograft model and cervical cancer specimens. Low levels of NHERF1 in cervical cancer specimens were found to associate with activation of cell proliferation and Wnt/β-catenin signaling by gene set enrichment analysis, and also were an independent predictive factor for worse prognosis of cervical cancer patients by Cox regression analysis. These findings demonstrate that NHERF1 inhibits Wnt signaling-mediated proliferation of cervical cancer via suppression of ACTN4, and NHERF1 downregulation may contribute to the progression of cervical cancer. These findings may also shed some lights for understanding the underlying mechanisms of cisplatin resistance and worse prognosis of HPV-inactive cervical cancer patients.

Mucinous colorectal adenocarcinoma (MCA) patients often a show high risk of malignant potential and a poorer survival rate. Given that the pathological feature and oncobiological characteristics of MCA are correlated with its abundant extracellular mucin2 (MUC2), we paid interest toward investigating the key factor that promotes MUC2 production exposure to highly-activated stem cell factor (SCF)/c-KIT signaling, which we believed to contribute to MCA formation. Long-term azoxymethane and dextran sodium sulfate treatment successfully induced MCA only in wild-type (WT) mice at week 37 and 43, while all c-kit loss-of-function mutant mice (Wadsm/m) developed non-MCA. Significantly, MUC2 and its key transcriptional factor Atonal homologue 1 (Atoh1) were remarkably expressed in MCA mice compared with non-MCA mice. Atoh1 was significantly elevated in colorectal cancer (CRC) cells stimulated by exogenous SCF or overexpressing c-KIT in vitro, while decreased by the blockage of SCF/c-KIT signaling with Imatinib. Furthermore, the maintained Atoh1 protein level was due to the inactive glycogen synthase kinase 3β (p-GSK3β) by virtue of the activated SCF/c-KIT-Protein Kinase B (AKT) signaling. Similar results were obtained from the ONCOMINE database and CRC patients. In conclusion, we suggested that SCF/c-KIT signaling promoted MUC2 production and MCA tumorigenesis by maintaining Atoh1 expression. Therefore, targeting the related key molecules might be beneficial for treating MCA patients.

// Jun Tan 1, 2 , Shu Yang 1, 2, 3 , Ping Shen 1, 3 , Haimei Sun 1, 2, 3 , Jie Xiao 1, 3 , Yaxi Wang 1, 2 , Bo Wu 1, 2, 3 , Fengqing Ji 1, 2, 3 , Jihong Yan 1 , Hong Xue 1 , Deshan Zhou 1, 2, 3 1 Department of Histology and Embryology, School of Basic Medical Sciences, Capital Medical University, Beijing 100069, P. R. China 2 Beijing Key Laboratory of Cancer Invasion and Metastasis Research, Beijing 100069, P. R. China 3 Cancer Institute of Capital Medical University, Beijing 100069, P. R. China Correspondence to: Deshan Zhou, e-mail: zhoudeshan2008@163.com Keywords: colorectal mucinous adenocarcinoma, C-kit, ETV4, murine model Received: March 24, 2015 Accepted: August 20, 2015 Published: September 02, 2015 ABSTRACT It was reported that the receptor tyrosine kinase (RTK) family often highly expressed in several mucinous carcinomas. In the present study, we established a murine model of colorectal mucinous adenocardinoma (CRMAC) by treating C57 mice [both wild type (WT) and loss-of-function c-kit mutant type (Wads −/− )] with AOM+DSS for 37 weeks and found that c-kit, a member of RTK family, clearly enhanced the tumor cell proliferation by decreasing p53 and increasing cyclin D1 through AKT pathway. Significantly, c-kit strongly promoted tumor cell invasiveness by increasing ETV4, which induced MMP7 expression and epithelial-mesenchymal transition (EMT) via ERK pathway. In vitro up- or down-regulating c-kit activation in human colorectal cancer HCT-116 cells further consolidated these results. In conclusion, our data suggested that the c-kit signaling obviously promoted proliferation and invasion of CRMAC. Therefore, targeting the c-kit signaling and its downstream molecules might provide the potential strategies for treatment of patients suffering from CRMAC in the future.

Diabetic gastroparesis is a common complication of diabetes mellitus (DM) that is characterized by decreased serum insulin and insulin-like growth factor-1 (IGF-1). Despite the fact that insulin treatment not glycemic control potently accelerated gastric emptying in type 1 DM patients, the role of insulin/InsR and IGF-1/IGF-1R signaling in diabetic gastroparesis remains incompletely elucidated. In the present study, type 1 DM mice were established and treated with insulin or Voglibose for 8 weeks. The gastric emptying was delayed from DM week 4 when the gastric InsR and IGF-1R were declined. Meanwhile, the gastric choline acetyltransferase (ChAT) was significantly reduced and the myenteric cholinergic neurones and their fibers were significantly diminished. The production of stem cell factor (SCF) was dramatically repressed in the gastric smooth muscles in DM week 6. TWereafter, interstitial cells of Cajal (ICC) were clearly lost and their networks were impaired in DM week 8. Significantly, compared with Voglibose, an 8-week treatment with insulin more efficiently delayed diabetic gastroparesis development by protecting the myenteric cholinergic neurones and ICC. In conclusion, diabetic gastroparesis was an aggressive process due to the successive damages of myenteric cholinergic neurones and ICC by impairing the insulin/InsR and IGF-1/IGF-1R signaling. Insulin therapy in the early stage may delay diabetic gastroparesis.

Extracellular toxins released by marine toxigenic algae into the marine environment have attracted increasing attention in recent years. In this study, profiling, characterization and quantification of extracellular toxin compounds associated with diarrhetic shellfish poison (DSP) in the culture medium of toxin-producing dinoflagellates were performed using high-performance liquid chromatography–high-resolution mass spectrometry/tandem mass spectrometry for the first time. Results showed that solid-phase extraction can effectively enrich and clean the DSP compounds in the culture medium of Prorocentrum lima (P. lima), and the proposed method achieved satisfactory recoveries (94.80%–100.58%) and repeatability (relative standard deviation ≤9.27%). Commercial software associated with the accurate mass information of known DSP toxins and their derivatives was used to screen and identify DSP compounds. Nine extracellular DSP compounds were identified, of which seven toxins (including OA-D7b, OA-D9b, OA-D10a/b, and so on) were found in the culture medium of P. lima for the first time. The results of quantitative analysis showed that the contents of extracellular DSP compounds in P. lima culture medium were relatively high, and the types and contents of intracellular and extracellular toxins apparently varied in the different growth stages of P. lima. The concentrations of extracellular okadaic acid and dinophysistoxin-1 were within 19.9–34.0 and 15.2–27.9 μg/L, respectively. The total concentration of the DSP compounds was within the range of 57.70–79.63 μg/L. The results showed that the proposed method is an effective tool for profiling the extracellular DSP compounds in the culture medium of marine toxigenic algae.

Background/aim Microsatellite instability (MSI) is a hallmark of genomic instability. In gastric cancer (GC), MSI phenotype is an important molecular subgroup that is often closely correlated with elevated mutation rates on the whole genome level. However, on a single gene level, it is still unknown whether the MSI status of a gene is correlated with the mutational profile of the gene itself. Materials and methods We analyzed intron MSI status and exon mutational profile of TP53 through short tandem repeat (STR) scanning and direct sequencing respectively in gastric cancers and their matched normal tissues. Results MSI status of the TP53 intron was significantly associated with the mutational profile of seven TP53 exon positions (p=0.0416), male patients (p=0.0095), and drinking (p=0.0474), and showed a mild correlation with longer survival time (p=0.0584) and increasing age (p=0.0611). Conclusion TP53 exons tended to mutation in the status of TP53 intron MSI.

Background/Aim: Overexpression of erythropoietin-producing hepatocellular A1 (EPHA1), a member of the EPH super family, is frequently observed in various cancer types. The dysregulated interaction of EPHA1 with its ligand Ephrin A1 has been linked to the progression of ovarian cancer (OC). However, the contribution of EPHA1 in the regulation of the aggressive properties of OC cells remains unknown. Materials and Methods: In this study we investigated the differential expression of EPHA1 in human OC cells. The EPHA1 gene was knocked-down using the CRISPR/Cas9 technique to evaluate its effect on the progressive properties of OC cells. Results: After EPHA1 was knocked-down using a CRISPR/CAS9 genomic editing system in OC cells (SKOV3 and COV504), we observed cell-cycle arrest at the G0/G1 phases in both OC cell lines. Knockdown of EPHA1 in the two OC cells inhibited their aggressive traits, including proliferation, invasion and migration, as well as improving their attachment to extracellular matrix. EPHA1 may play a role in OC through its regulation of multiple signaling pathways, such as matrix metalloproteinase-2 (MMP2), extracellular signal-regulated kinase 2 (ERK2) and proto-oncogene c-MYC. Conclusion: EPHA1 may promote the aggression of some OC cells and, thus, be considered a potential therapeutic target for the treatment of malignant OC.

Claudin-3 is a major protein of tight junctions (TJs) in the intestinal epithelium and is critical for maintaining cell-cell adhesion, barrier function, and epithelium polarity. Recent studies have shown high claudin-3 levels in several solid tumors, but the regulation mechanism of claudin-3 expression remains poorly understood. In the present study, colorectal cancer (CRC) tissues, HT-29 and DLD-1 CRC cell lines, CRC murine model (C57BL/6 mice) and c-kit loss-of-function mutant mice were used. We demonstrated that elevated claudin-3 levels were positively correlated with highly expressed c-kit in CRC tissues based upon analysis of protein expression. In vitro, claudin-3 expression was clearly increased in CRC cells by overexpressed c-kit or stimulated by exogenous recombinant human stem cell factor (rhSCF), while significantly decreased by the treatment with c-kit or c-Jun N-terminal kinase (JNK) inhibitors. Chromatin immunoprecipitation (ChIP) and luciferase reporter assay showed that SCF/c-kit signaling significantly promoted activator protein-1 (AP-1) binding with CLDN-3 promoter and enhanced its transcription activity. Furthermore, decreased expression of claudin-3 was obtained in the colonic epithelium from the c-Kit loss-of-function mutant mice. In conclusion, SCF/c-kit-JNK/AP-1 signaling pathway significantly promoted claudin-3 expression in colonic epithelium and CRC, which could contribute to epithelial barrier function maintenance and to CRC development.

MiR-34c is considered a potent tumour suppressor because of its negative regulation of multiple target mRNAs that are critically associated with tumorigenesis and metastasis. In the present study, we demonstrated a novel target of miR-34c, KITLG, which has been implicated in colorectal cancer (CRC). First, we found a significant negative relationship between miR-34c and KITLG mRNA expression levels in CRC cell lines, including HT-29, HCT-116, SW480 and SW620 CRC cell lines. In silico analysis predicted putative binding sites for miR-34c in the 3' untranslated region (3'UTR) of KITLG mRNA. A dual-luciferase reporter assay further confirmed that KITLG is a direct target of miR-34c. Then, the cell lines were infected with lentiviruses expressing miR-34c or a miR-34c specific inhibitor. Restoration of miR-34c dramatically reduced the expression of KITLG mRNA and protein, while silencing of endogenous miR-34c increased the expression of KITLG protein. The miR-34c-mediated down-regulation of KITLG was associated with the suppression on proliferation, cellular transformation, migration and invasion of CRC cells, as well as the promotion on apoptosis. Knockdown of KITLG by its specific siRNA confirmed a critical role of KITLG down-regulation for the tumour-suppressive effects of miR-34c in CRC cells. In conclusion, our results demonstrated that miR-34c might interfere with KITLG-related CRC and could be a novel molecular target for CRC patients.

Dopaminergic (DA) neuron therapy has been established as a new clinical tool for treating Parkinson's disease (PD). Prior to cell transplantation, there are two primary issues that must be resolved: one is the appropriate seed cell origin, and the other is the efficient inducing technique. In the present study, human umbilical cord blood-derived mesenchymal stem cells (hUCB-MSCs) were used as the available seed cells, and conditioned medium from human amniotic epithelial cells (ACM) was used as the inducing reagent. Results showed that the proportion of DA neuron-like cells from hUCB-MSCs was significantly increased after cultured in ACM, suggested by the upregulation of DAT, TH, Nurr1, and Pitx3. To identify the process by which ACM induces DA neuron differentiation, we pretreated hUCB-MSCs with k252a, the Trk receptor inhibitor of brain-derived neurotrophic factor (BDNF) and nerve growth factor (NGF), and found that the proportion of DA neuron-like cells was significantly decreased compared with ACM-treated hUCB-MSCs, suggesting that NGF and BDNF in ACM were involved in the differentiation process. However, we could not rule out the involvement of other unidentified factors in the ACM, because ACM + k252a treatment does not fully block DA neuron-like cell differentiation compared with control. The transplantation of ACM-induced hUCB-MSCs could ameliorate behavioral deficits in PD rats, which may be associated with the survival of engrafted DA neuron-like cells. In conclusion, we propose that hUCB-MSCs are a good source of DA neuron-like cells and that ACM is a potential inducer to obtain DA neuron-like cells from hUCB-MSCs in vitro for an ethical and legal cell therapy for PD. © 2013 Wiley Periodicals, Inc.

Hypermethylation often leads to gene silencing; however, the mechanism responsible for the low expression resulting from hypermethylation of the tumor suppressor gene Kelch-like ECH-associating protein 1 (Keap1) in human lung cancer cell lines remains unclear. In this study, using promoter deletion and site mutagenesis assays, we determined that one transcription factor stimulating protein-1 (Sp1) regulatory element in the Keap1 promoter region was important for the transcription of Keap1 in A549 cells. We demonstrated that the transcription factor Sp1 can directly bind to this element in the normal bronchial epithelial BEAS-2B cell line but not in A549 cells, as assessed with chromatin immunoprecipitation (ChIP). EMSAs and supershift assays also showed that CpG island methylation could abrogate Sp1 binding to the Keap1 promoter. Moreover, Keap1 mRNA decreased by 50% after the knock-down of Sp1 with siRNA in BEAS-2B cells, whereas the over-expression of Sp1 led to a dramatic increase in Keap1 promoter activity. The treatment of A549 cells with 5-aza-2'-deoxycytidine restored the binding of Sp1 to the promoter and Keap1 expression. Our results indicate that Sp1 is essential for Keap1 expression and that promoter methylation blocks Sp1 binding in A549 cells. These results demonstrate that hypermethylation may act as an epigenetic gene silencing mechanism, i.e., the inhibition of Sp1 binding to the hypermethylated Keap1 promoter in lung cancer cells, which suggests new approaches to lung cancer treatment.

Normal aging is commonly associated with decreased cognitive functions, which could be conspicuously alleviated by enriched environment (EE) with physical, social, and sensory stimuli, suggesting that aging brain still has intriguing plasticity. Multiple researches have been carried out to explore the structural and the molecular changes in aging brain, which would be considered for evidences that EE regulated brain plasticity. Because there is no significant neuron loss in aging cerebral cortex and the white matter is crucial for cognitive functions, this review focused on the age-related white matter changes and the effects of EE on aged white matter. Data from our stereology laboratory revealed that age-related spatial memory declines had more to do with white matter alterations, which were due to marked demyelination and loss of oligodendrocytes in the white matter. We also demonstrated that EE recovered spatial memory impairment and increased white matter volume by promoting marked remyelination in aged brain. This review approached the issue that EE might contribute to normal aging and be beneficial for those suffering from demyelinated diseases.

The anticancer effects of artesunate (ART) have been well documented. However, its potential against skin cancer has not been explored yet. Herein we reported that 60 μmol/l ART effectively inhibited A431 (human epidermoid carcinoma cells) growth but not that of HaCaT (normal human keratinocyte cells). Our results revealed that ART induced cell cycle arrest at G0/G1 phase through the downregulation of cyclin A1, cyclin B, cyclin D1, Cdk2, Cdk4, and Cdk6. This correlated with the upregulation of p21 and p27. The 5-bromodeoxyuridine incorporation assay also indicated that ART treatment reduced DNA synthesis in a time-dependent manner. Furthermore, ART induced mitochondrial apoptosis, as evidenced by annexin V/propidium iodide staining and western blot analysis. Interestingly, ART-induced apoptosis diminished under iron-deficient conditions but intensified under iron-overload conditions. Taken together, these findings demonstrated the potential of ART in treating skin cancer through the induction of G0/G1 cell cycle arrest and iron-mediated mitochondrial apoptosis and supported further investigations in other test systems.

Although it is well known that the reduction of interstitial cells of Cajal (ICCs) is associated with several gastrointestinal motility disorders in clinic, it is unknown whether the mature ICCs still have an active plasticity in adult mammals. This study focused on the issues of the reduction of ICCs during Imatinib administration and the recovery of ICCs following drug withdrawal in the small intestine of adult guinea pigs. ICCs were revealed by immunofluorescence on whole mount preparations with anti-Kit, alpha-smooth muscle actin, (alpha-SMA), and 5-bromo-2'-deoxyuridine (BrdU) antibodies. Moreover, the occurrence of apoptosis was also assayed. Imatinib treatment led to a gradual reduction of ICCs in number around the myenteric plexus and deep muscular plexus, which was dependent on the time but no apoptosis of ICCs was detected with the TUNEL method. During Imatinib treatment, some ICC-like cells were double labeled for Kit and alpha-SMA and a few ICC-like cells were only stained with alpha-SMA. When Imatinib was discontinued, the number of ICCs recovered to normal within 32 days. During this time, some proliferating ICCs were demonstrated by double labeling with Kit and BrdU antibodies. Our results indicated that Kit signaling was essential for the maintenance of survival and proliferation of the mature ICCs in the small intestine of adult guinea pigs. Moreover, ICCs might transdifferentiate to a type of alpha-SMA(+) cells, perhaps a phenotype of smooth muscle cells, when there is a loss-of-function of Kit.

This study aimed at evaluating whether apoptosis of interstitial cells of Cajal (ICC), smooth muscle cells (SMC), and enteric neurons was involved in a guinea pig model of intestinal ischemia and reperfusion injury. The small intestinal segments were resected at either 6 (I60/R6h) and 12 h (I60/R12h) or 7 (I60/R7d) to 14 (I60/R14d) days after 60 min intestinal ischemia in the adult guinea pigs and studied by immunohistochemistry with anti-Kit, 5-bromo-2'-deoxyuridine (BrdU), alpha-smooth muscle actin, vimentin, and beta-tublin III antibodies. Also, apoptosis was tested by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) method. In the I60/R12h injury, there was a approximately 50% decrease of Kit+ cells in cell numbers at the level of myenteric plexus and a number of Kit-/vimentin-positive cells were labeled by TUNEL. Also, a few SMC and enteric neurons were TUNEL positive. The Kit+ ICC recovered to normal and a number of Kit-/BrdU-double-positive cells were observed in the I60/R14d group. Our results indicated that the intestinal I/R injury could lead to apoptosis of ICC, SMC, and enteric neurons which may contribute to the gastrointestinal motility disorders, and proliferation was involved in the recovery of ICC.

This paper aimed at investigating the alterations in interstitial cells of Cajal (ICCs) in the murine small intestine from 0-day to 56-day post-partum (P0-P56) by immunohistochemistry. The Kit+ ICCs, which were situated around myenteric nerve plexus (ICC-MY) formed a loose cellular network at P0 which changed into an intact one before P32. The density of ICC-MY increased from P0 to P12, and then decreased until P32. In contrast, the estimated total amount increased more than 15-fold at P32 than that at P0. Some Kit+/BrdU+ cells were observed at 24 h after one BrdU injection to the different-aged mice, and the number decreased from P2 to P24 and vanished at P32. Actually a few Kit+/BrdU+ cells can be observed at 1 h after one BrdU injection at P10, and the amount doubled at 24 h along with paired Kit+/BrdU+ cells. A number of BrdU+ ICCs were also labeled with CD34, CD44 and insulin-like growth factor I receptor. About 65% ICCs were BrdU+ at P32 after daily BrdU injection from P0. Our results indicate that an age-dependent proliferation is involved in the postnatal development of ICC-MY which increase greatly in cell numbers and proliferative ICCs may originate from ICCs progenitor cells.

Low expression of the oxidative stress sensor Keap1 is thought to be involved in carcinogenesis. However, the mechanisms responsible for inactivation of the Keap1 gene remain unknown. We investigated Keap1 expression using RT-PCR and found that it was downregulated in lung cancer cell lines and tissues when compared with a normal bronchial epithelial cell line. Treatment with 5-Aza-2'-deoxycytidine restored Keap1 expression in lung cancer cell lines, indicating the silencing mechanism to be promoter methylation. Moreover, we evaluated cytosine methylation in the Keap1 promoter and demonstrated that the P1 region, including 12 CpG sites, was highly methylated in lung cancer cells and tissues, but not in normal cells. Importantly, we found evidence that three specific CpG sites (the 3rd, 6th, and 10th CpGs of P1) might be binding sites for proteins that regulate Keap1 expression. Thus, our results suggest for the first time that Keap1 expression is regulated by an epigenetic mechanism in lung cancer.

Although osteonecrosis of the femoral head is a known primary limitation of long-term or high-dose clinical administration of glucocorticoids, the mechanisms underlying this side effect remain unclear. Hypoxia is an important biological state under numerous pathological conditions. In this study, we investigated glucocorticoid-induced osteonecrosis under hypoxic conditions in the MC3T3-E1 osteoblast cell line using a cell cytotoxicity assay, flow cytometry, and western blotting. 6α-Methylprednisolone sodium succinate (MPSL) more effectively induced apoptosis and G0/G1 arrest of MC3T3-E1 osteoblasts under hypoxic conditions than under normoxic conditions. Correspondingly, MPSL more effectively upregulated cellular levels of cleaved caspase 3, p53, and its target p21, and downregulated cyclin D1 levels in hypoxia. Moreover, overexpression of Akt abrogated the MPSL activation of p53, p21, and cleaved caspase 3 and the attenuation of cyclin D1 expression and rescued osteoblasts from MPSL-induced cell cycle arrest and apoptosis, indicating that phosphatidylinositol 3-kinase (PI3K)/Akt signaling might play an essential role in MPSL-induced inhibition of osteoblasts. Furthermore, the suppression of PI3K/Akt signaling and upregualtion of cellular p85α monomer levels by MPSL were more pronounced under hypoxic conditions than under normoxic conditions. Finally, we found that the enhancement of the effects of MPSL under hypoxic conditions was attributed to hypoxia-upregulated glucocorticoid receptor activity. In conclusion, our results demonstrate that MPSL, a synthetic glucocorticoid receptor agonist, promotes the level of p85α and inhibits PI3K/Akt signaling to induce apoptosis and cell cycle arrest in osteoblasts, and that this effect is enhanced under hypoxic conditions.

To investigate whether Na(+)-K(+)-2Cl(-) cotransporter (NKCC2) is expressed in the mouse distal colonic epithelia and whether it is regulated by vasopressin in the colon.The mRNA expression of NKCC2 in the mouse colonic mucosa was examined by reverse transcription-polymerase chain reaction. NKCC trafficking in the colon stimulated by 1-D-amino(8-D-arginine)-vasopressin (dDAVP) infusion (10 ng/mouse, intraperitoneal injection ) within 15 min, 30 min and 1h was investigated by laser confocal scanning microscopy. Total and membrane NKCC2 expression in the colonic mucosa from control and dDAVP-treated mice was detected by Western blotting. Short circuit current method was performed to determine regulation of NKCC2 by vasopressin in the colon.NKCC2 was predominantly located in the apical region of the surface of the distal colonic epithelia; by comparison, a large amount of NKCC1 was distributed in the basolateral membrane of the lower crypt epithelia of the mouse distal colon. Short-term treatment with dDAVP, a V2-type receptor-specific vasopressin analog, induced NKCC2 re-distribution, i.e., NKCC2 traffics to the apical membrane after dDAVP stimulation. In contrast, no obvious NKCC1 membrane translocation was observed. Western blotting results confirmed that membrane NKCC2 had significantly higher abundance in the dDAVP-treated mouse colonic mucosa relative to that in the untreated control, which is consistent with our immunostaining data. Moreover, the short-circuit current method combined with a NKCC2 inhibitor demonstrated that NKCC2 was also activated by serosal vasopressin in isolated distal colonic mucosa.Our results provide direct evidence that vasopressin also plays an important role in the colonic epithelia by stimulating NKCC2 trafficking to the apical membrane and inducing NKCC2-mediated ion transport.

Insulin-like growth factor-I (IGF-I) and its receptor (IGF-IR) have tremendous trophic effects on the central, peripheral and enteric neurons. The loss of IGF-IR contributes to the development of diabetic gastroparesis. However, the nature and the function of the IGF-IR(+) cells in the gastric myenteric plexus remain unclear. In this study, anti-ChAT, anti-S100β or anti-c-KIT antibodies were used to co-label IGF-IR(+) cells and neurons, glial cells or interstitial cells of Cajal (ICCs), respectively. We also generated type 1 diabetic mice (DM) to explore the influence of impaired IGF-I/IGF-IR in the myenteric neurons. Results showed that IGF-IR was expressed in the epithelium, smooth muscles and myenteric plexi of the mouse stomach. Most of the IGF-IR(+) cells in the myenteric plexi were ChAT(+) cholinergic neurons, but not enteric glial cells and there were more IGF-IR(+) neurons and fibers in the gastric antrum than in the corpus. The IGF-IR(+)/ChAT(+) neurons and ICCs were closely juxtaposed, but distinctly distributed in the myenteric plexus, indicating a possible role for the IGF-IR(+)/ChAT(+) neurons in the mediation of gastric motility through ICCs. Moreover, the decrease of IGF-IR and cholinergic neurons in the myenteric plexi and smooth muscles of DM mice suggested that IGF-I/IGF-IR signaling might play a role in neuron survival and neurite outgrowth, as well as stem cell factor (SCF) production, which is required for the development of ICCs. Our results provide insights into the effects of IGF-I/IGF-IR signaling on the development of gastrointestinal motility disorders.

In actinomycin D (AD)-induced apoptosis, caspase-3 activation and DNA cleavage in human megakaryoblastic leukemia CMK-7 cells were greatly accelerated by tubulin and actin polymerization inhibitors [e.g., colcemid (CL) and cytochalasin D (CD), respectively], but the acceleration was not found with Taxol or phalloidin. A decrease in mitochondrial transmembrane potential, release of cytochrome c into the cytosol, and cleavage of procaspase-9 to its active form preceded the activation of caspase-3 and, moreover, all of these events began earlier and/or proceeded faster in cells treated with AD plus CL or CD than in cells treated with AD only. These results suggest that cytoskeletal disruption in the apoptotic cells promotes damage of the mitochondrial membrane, resulting in the enhanced release of cytochrome c necessary for the activation of caspase-9 that initiates the caspase cascade. On the other hand, apoptotic bodies were rapidly formed from cells treated with AD and CL, but were suppressed when treated with AD and CD. Intracellular membranes and the actin system were reorganized to surround the nuclear fragments in the AD- and CL-treated cells, but such a membrane system was not formed in the presence of CD, implying that the apoptotic bodies are formed via reorganization of intracellular membranes under regulation by actin polymerization. Thus, the cytoskeletal change in CMK-7 cells has a strong effect on the early biochemical process as well as on the later morphologic process in AD-induced apoptosis.

Influenza virus hemagglutinin is a paradigm for biological membrane fusion mechanisms. Among the most important recent progresses in this field are “spring-loaded mechanism” based on a coiled-coil of α-helices and “microprotrusion of lipid bilayer” which was found through a quick-freezing electron microscopic observation. Possible molecular architectures of membrane fusion intermediates induced by hemagglutinin are discussed.

The development and maintenance of interstitial cells of Cajal (ICC) are closely associated with SCF/KIT signal activity. In this study, we evaluate the distribution of ICC in KIT distal kinase domain mutant mice (Wads) and determine whether the loss-of-function mutations in KIT easily lead to gastrointestinal (GI) disorders. ICC were examined by anti-KIT immunohistochemistry and western blotting. The GI microstructure of wild-type (WT) and Wads mice in normal intestines and incomplete intestinal obstruction was evaluated by hematoxylin and eosin staining. The results in Wads(m/m) mice were as follows. Myenteric ICC were obviously decreased in the stomach and colon and were totally absent in the small intestine. Intramuscular ICC were nearly absent in the stomach and irregularly distributed in the colon. Moreover, the smooth muscle thickness of the small intestine was increased 1.3-fold in Wads(m/m), compared to WT and Wads(m/+) mice and the diameter of the intestinal lumen was also enlarged in Wads(m/m) mice. When constructing an incomplete intestinal obstruction model, the extent of distention involved was greater in Wads mice (1.6-fold in Wads(m/+) mice and 1.8-fold in Wads(m/m) mice vs. WT mice). Meanwhile, the intestinal lumen expansion and decrease in ICC were more pronounced in Wads mice than in WT mice. Our results suggest that the KIT distal kinase domain mutation leads to an ICC loss in a subtype and location-specific pattern in Wads(m/m) mice. The injury of the KIT signaling in mutant mice results in more serious pathological manifestations after being exposed to pathogenic factors.

The purpose of this study was to investigate the mechanism by which magnolol treatment prevents lipopolysaccharide (LPS)-induced septic dysmotility in mice. Sepsis was induced by intravenous tail vein injection of LPS (4 mg/kg body weight). Animals were divided into three groups: the magnolol-treated septic group, the placebo-treated septic group, and the control group. Intestinal transit and circular smooth muscle contraction were measured 12 h after LPS injection, and immunocytochemisty was performed to study the morphology of interstitial cells of Cajal (ICCs). Stem cell factor (SCF) expression and c-kit phosphorylation were determined by Western blot analysis, and the mRNA levels of inducible NO synthase (iNOS) were determined by RT-PCR. Nitric oxide (NO) content, superoxide dismutase (SOD) activity, and malondialdehyde (MDA) concentration were detected using commercial kits. Intestinal transit and muscular contractility were significantly lower in the LPS-treated group than in the control group. Immunocytochemical experiments showed that the total number of ICCs, and the total and average lengths of the ICC processes were significantly decreased in the LPS-treated group compared with those in the control group. In LPS-treated animals, magnolol pretreatment significantly accelerated intestinal transit, increased circular muscle contraction, and prevented ICC morphology changes. Phosphorylation of c-kit and expression of SCF were significantly downregulated in LPS-treated animals compared with control animals. Magnolol pretreatment prevented sepsis-induced decreases in c-kit phosphorylation and SCF expression in LPS-treated animals. Magnolol pretreatment prevented the sepsis-induced increase in NO concentration, iNOS expression, and MDA concentration, and decrease in SOD activity in LPS-treated animals. Our results suggest that magnolol treatment prevents sepsis-induced intestinal dysmotility by regulating SCF/c-kit and NO signaling to maintain functional ICCs.

We have reported that human umbilical cord blood-derived mesenchymal stem cells (hUCB-MSCs) are capable of differentiating into dopaminergic (DA) neuron-like cells upon being induced by amniotic epithelial cells (AECs). However, what factor(s) is involved in the differentiation process has not been explored out thoroughly. Because pleiotrophin (PTN) is known to exert important trophic effects on DA neurons, in the present study, we investigated whether PTN is released by AECs and whether it is involved in the differentiation of hUCB-MSCs into DA neuron-like cells. The expression and secretion of PTN by AECs were detected by immunofluorescence, RT-PCR and ELISA. The hUCB-MSCs were isolated and treated with AEC-conditioned medium (ACM) or recombinant human PTN. Compared to the controls, a higher proportion of treated cells differentiated into DA neuron-like cells, indicated by the increased expression of TH and DAT and the increased dopamine content. These results indicate that PTN released by AECs acts as a synergetic factor with other neurotrophic factors and is involved in the differentiation of hUCB-MSCs into DA neuron-like cells. We suggest that ACM, which contains PTN and other neurotrophic factors, could potentially be used as an agent to promote the differentiation of DA neuron-like cells from hUCB-MSCs for cell therapy of Parkinson's disease without creating legal or ethical issues.

Objective Cisplatin is a widely used chemotherapeutic agent in the treatment of cancers in clinic; but it often induces adverse effects on ovarian functions such as reduced fertility and premature menopause. Mesna could attenuate the cisplatin-induced ovarian damages; however, the underlying mechanism is still unknown. This study aimed to figure out the underlying mechanism of the protection of mesna for ovaries against cisplatin therapy in cancers. Methods We performed female adult Sprague-Dawley rats into normal saline control (NS), low-dose cisplatin (CL), high-dose cisplatin (CH), CL plus mesna (CL+M), and CH plus mesna (CH+M) groups and detected anti-Mullerian hormone (AMH)-positive follicle, oxidative stress status and anti-oxidative capability in ovaries. Results AMH-positive follicles were significantly decreased after cisplatin administration, which was significantly reversed when mesna was co-administered with cisplatin. The end product of lipid peroxidation, malondialdehyde (MDA), was significantly increased, but the anti-oxidative enzymatic activity of superoxide dismutase (SOD) and glutathione (GSH) were significantly decreased in cisplatin groups when compared with NS group. In contrast, after co-administration of cisplatin with mesna, MDA was significantly decreased whereas the activity of SOD and the concentration of GSH were increased. Moreover, mesna did not decrease the anti-tumor property of cisplatin in HePG2 cell lines. Conclusion Cisplatin damages the granulosa cells by oxidative stress to deplete the ovarian reserve and mesna could protect ovarian reserve through anti-oxidation. These results might highlight the mechanism of the protection of mesna for ovarian reserve and open an avenue for the application of mesna as a protective additive in cisplatin chemotherapy in clinical practise.

HCN2 channels are involved in the spontaneous rhythmic activities of some CNS neurons and act by generating I(f) current. The gastrointestinal (GI) tract is known to be capable of spontaneous rhythmic activity; however, the possible role of HCN2 channels in this organ has not yet been elucidated. This study investigated the distribution of HCN2-positive cells in the mouse GI tract using immunohistochemistry. To identify the nature of these HCN2 cells, anti-ChAT and anti-Kit antibodies were used to co-label neurons and the interstitial cells of Cajal (ICCs), respectively. Additionally, differences in the distribution of HCN2-positive cells within the GI tract were also analyzed. Our results showed that HCN2 channels were mainly located within the myenteric neurons of the enteric nervous system in the GI tract. Double-staining revealed that HCN2-positive neurons were labeled by ChAT, indicating that these HCN2-positive cells are also cholinergic neurons. Although the HCN2-positive cells were not stained by the anti-Kit antibody, their processes were in close proximity to ICCs around the myenteric plexus region. Moreover, several differences in the distribution of HCN2 in the stomach, small intestine and colon were partly consistent with the regional differences in the spontaneous rhythmic activities of these organs. Basing on the role HCN2, we suggested that HCN2 channels facilitate the release of Ach from cholinergic neurons to affect the GI peristalsis by acting on M receptors on the ICCs. However, the HCN2 channels are not directly involved in spontaneous slow-wave initiation by ICCs.

Ultraviolet radiation A (UVA)-induced oxidative stress is recognized as an important factor in the development of skin carcinogenesis. Resveratrol is demonstrated to possess remarkable antioxidant activity in the organism. The aim of this study was to investigate the protective role of resveratrol in human keratinocytes (HaCaT) against UVA-induced oxidative damage and the possible mechanism of the translocation of NF-E2-related factor-2 (Nrf2) into the nucleus. The HaCaT cells were UVA-irradiated and the effects of resveratrol on cell viability, reactive oxygen species generation and membrane-lipid peroxidation were measured. The proteins and mRNA of Nrf2 and Kelch-like-ECH-associated protein 1 (Keap1) were determined by immunofluorescence staining, Western blot and quantitative PCR, respectively. UVA exposure led to a decrease in viability and an increase in reactive oxygen species generation in HaCaT cells. Resveratrol could effectively increase the viability of HaCaT cells after UVA exposure and protect them from UVA-induced oxidative stress. Moreover, resveratrol increased the level of Nrf2 protein and facilitated Nrf2 accumulation in the nucleus; as a result, the activity of antioxidant enzymes was also upregulated. The main finding was that Keap1 protein, a repressor of Nrf2 in the cytoplasm, was clearly decreased by resveratrol treatment 12h and beyond though the level of Keap1 mRNA still increased. Our results suggest that resveratrol can degrade Keap1 protein and facilitate Nrf2 accumulation in the nucleus, thereby protecting HaCaT cells from UVA-induced oxidative stress. Resveratrol could be a more useful natural medicine for the protection of epidermal cells from UVA-induced damage.

J. Neurochem. (2010) 114, 1039–1048. Abstract Our previous studies show that group III secreted phospholipases A2 (sPLA2s III) induces extensive neuronal apoptosis in brain cortical cultures. However, the molecular mechanisms underlying sPLA2 III-induced neuronal injury/death are still unknown. Also it is not clear whether hypoxic pre-conditioning (HPC) is able to protect neurons from the sPLA2 III insult. In this report, we demonstrate that sPLA2 III significantly decreased production of Bcl-xl and the ratio of Bcl-xl/Bax, and increased expression of Bax, cleaved caspase 3, and cleaved α-Fodrin in primary neuronal culture. HPC prevented the sPLA2 III-induced decreases in production of Bcl-xl and the ratio of Bcl-xl/Bax, and increases in expression of Bax, cleaved caspase 3, and α-Fodrin. However, the HPC-produced neuronal protection was eliminated or attenuated by AG490, rapamycin, and STAT3 shRNA. Our results suggest that sPLA2 III-induced neuronal apoptosis is likely because of its alterations in expression and activity of Bcl-xl, Bax, caspase 3, and its target gene fodrin; and that HPC-produced neuroprotection against the sPLA2 III toxicity is mediated via JAK-STAT signal pathways that regulate the expression of Bcl-xl, Bax, and cleaved caspase 3 in cultured cortical neurons.

It was reported that a signaling pathway of platelet-derived growth factors (PDGFs) and their receptors (PDGFRs) played a critical role in the developing gut of mice. Overexpression of the PDGFR-alpha gene in gastrointestinal stromal tumors (GISTs) indicated that parts of tumor cells originated from PDGFR-alpha-positive cells, but a more detailed distribution of PDGFR-alpha and possible role in the adult mammalian gut are still unclear. In the present study, we examined the expression of both PDGF-AA and its receptor PDGFR-alpha in the gastrointestinal (GI) tract of adult guinea pigs using western blotting and immunohistochemistry. PDGF-AA-immunoreactive cells were mainly distributed in the mucosal epithelium of the stomach, small intestine, and large intestine. Only a few PDGF-AA-positive cells were seen in the longitudinal muscle layer of the large intestine. In contrast, PDGFR-alpha-positive cells were widely distributed throughout the GI tract, including the lamina propria, muscular layer, and subserosa. Double staining showed that the distribution of the PDGFR-alpha-positive cells in the muscular layer were similar to those of the interstitial cells of Cajal (ICCs), and they were associated with ICCs and enteric nerves, but no double-labeled cells were observed by anti-PDGFR or Kit antibody. It was noted that PDGFR-alpha-positive cells were also stained with a vimentin monoclonal antibody. Based on the double staining and morphological features, we consider the PDGFR-alpha-positive cells belong to a subtype of fibroblast. Our results not only provide a roadmap for understanding the function of the PDGF/PDGFR signaling pathway in both normal adult mammals and during gut injury and repair but also might help in understanding the growth and development of GISTs in the clinic.

It has been suggested that interstitial Cajal-like cells (ICLC) may be involved in the spontaneous rhythmic electrical activities of the extrahepatic bile duct system. The present study investigated the distribution and characteristics of ICLC, which are immunopositive for CD117/ Kit receptor tyrosine kinase, using immunohistochemistry employing a monoclonal antibody raised against CD117/Kit on whole-mount preparations. The Kit-positive ICLC were examined using confocal laser scanning microscopy or fluorescence microscopy. ICLC, immunoreactive for Kit, were pleiomorphic and/or spindle-shaped cells with a few bipolar processes and distributed in the smooth muscle layers of the gallbladder and bile duct system. They were scattered in the hepatic duct, cystic duct and gallbladder as well as in the upper part of the common bile duct. The ICLC gradually increased in number and formed a completed cellular network in the lower part of the common bile duct and ampulla. The numbers of ICLC in the ampulla were similar to that of the duodenum and significantly much greater in number than in the gallbladder and bile ducts. The density of the ICLC in the common bile duct was significantly higher than that of other bile ducts. Our results suggested that the ICLC might contribute to the regulation of the spontaneous rhythmic contraction and development of motility disorders of the bile duct system.

Surgical manipulations of the gastrointestinal (GI) tract usually lead to loss of interstitial cells of Cajal (ICCs). The present study prepared to investigate whether ICCs can regenerate and restore their normal distribution up to 5 months after semitransection and end-to-end anastomosis of small intestines of adult guinea pigs. The segments of anastomosis were studied by immunohistochemistry with anti-KIT, 5-bromo-2'-deoxyuridine (BrdU), stem cell factor (SCF), and neurofilament 200 antibodies and also by transmission electron microscopy (TEM). At early stage, intestinal surgery led to intestinal wall impairment and ICCs loss, and ICCs near the site of anastomosis gradually increased in numbers. About 150 days postoperation, the distribution of ICCs and the microstructure of intestinal wall appeared to be similar with those of the control. By double immunostaining with BrdU and KIT antibodies, a number of proliferated ICCs were seen near the site of transection/anastomosis. Furthermore, KIT ligand, SCF, was mainly observed in the smooth muscle cells (SMCs), which are located close to ICCs. TEM observation revealed a number of immature and mature ICCs in this region. Our results indicated that ICCs could regenerate and restore their normal distribution after intestinal surgery and SMCs might be involved in the regenerated events of ICCs in the adult guinea pig GI tract.

Pulmonary arterial smooth muscle cells (PASMC) were divided into a normoxic group (N), 2, 8 and 12 h hypoxic groups (H2, H8 and H12) and an AG490 plus 8 h hypoxic group (AG490). The expression of JAK1, JAK2, JAK3 and TYK2 mRNA was analyzed by reverse transcription-polymerase chain reaction (RT-PCR). STAT1 and STAT3 protein expressions were determined by Western blotting. The results showed that the levels of JAK1, JAK2 and JAK3 mRNA did not change significantly in the N group but were increased after 2 h exposure to hypoxia. JAK1, JAK2 and JAK3 mRNA expressions peaked at 8 h. It decreased at 12 h but remained above those in the N group. TYK2 mRNA was not found in either hypoxic or normal PASMC. The phospho-STAT1 and -STAT3 protein levels increased after 2 h exposure to hypoxia. They were about 2.8 and 4.1 times the N group, respectively, after 8 h. They decreased at 12 h but remained above those in the N group. AG490 inhibited phospho-STAT3 protein expression by about 25.5% at 8 h but did not block the expression of phospho-STAT1 protein. These findings suggest that hypoxia induces the expression of JAK1, JAK2, JAK3 and phospho-STAT1 and -STAT3 in PASMC. Hypoxia activates the JAKs-STATs signaling pathway, which may participate in the pathogenesis of hypoxic PASMC injury.

The vesicles composed of synthetic phytanyl-chained glycolipid and natural sulfoquinovosyldiacylglycerol at 9:1 molar ratio were successfully applied to functional reconstitution of photosystem II complex (PS II) from a thermophilic cyanobacterium. The synthetic glycolipid employed was one of our model archaeal diether lipids, 1,3-di- O -phytanyl-2- O -(β-D-maltotriosyl)glycerol. The light-induced oxygen-evolving activity of PS II reconstituted in the glycolipid vesicles was approximately 6-fold higher than that reconstituted in several phosphatidylcholine vesicles. The present results reveal the first evidence that a well-designed synthetic glycolipid is effective for the functional reconstitution of complicated and labile membrane protein complexes, such as PS II.

Human megakaryoblastic leukemia Meg-01 cells were attached to fibronectin (FN)-coated substratum, on which remarkable spreading and cytoplasmic elongation was induced by treatment with a protein kinase inhibitor, staurosporine (stp). This effect was inhibited by RGDS and was also not seen on FN-lacking substratum. The extended cytoplasm had swollen terminals and nodes, which contained GpIIb and β-thromboglobulin, occasionally included α granules, and tended to form particles (2–5 μm) after rupture of the narrowed cytoplasm. Among other protein kinase modulators tested, only K252a promoted the elongation, while calphostin, herbimycin, TPA, and calyculin suppressed it. The cells began to migrate soon after addition of stp, with attachment to the substratum held at some sites during the migration. This tethered movement seemed to cause the cytoplasmic elongation and the rupture into particles. The elongation was retarded by pretreating the cells with cytochalasin A and Clostridium C3 toxin but not with demecolcine. Actin microfilaments in the stp-treated Meg-01 cells accumulated in the filopodia and periphery of the extended cytoplasm, in which vinculin was colocalized as adhesion plaques. The microtubules were longitudinally oriented through the cytoplasmic extension and showed no ring profile in the nodes and particles. Thus, stp in the presence of FN appears to stimulate reorganization of actin-based cytoskeleton and formation of focal contacts in Meg-01 cells. This leads to the activation of cell adhesion and motility, and then cytoplasmic elongation and rupture into particles. J. Cell. Physiol. 179:179–192, 1999. © 1999 Wiley-Liss, Inc.

The apoptosis induced by actinomycin D (AD) in human mega-karyoblastic leukemia CMK-7 cell line is accelerated by microtubule poisons such as colcemid. We previously reported that the cell fragmentation in this apoptosis was suppressed by cytochalasin D (CD). 1 Here, we report that the AD-induced apoptosis is also accelerated by CD. The caspase-3 activation and DNA cleavage reached the maximum at least 10 hr earlier in the presence of CD than without this actin polymerization inhibitor. Decrease in organelle membrane potential, cytochrome c release from mitochondria, and cleavage of procaspase-9 to its active form began to appear prior to the caspase-3 activation. Apaf-1 was detected in the cytosol. These results suggest that the autocatalytic activation of caspase-9 starts the usual caspase cascade, and that the determinant, cytochrome c liberation, is promoted by interfering with the actin system. The cell surface F-actin disappeared by the AD-CD treatment and instead broad actin fibers appeared in the nucleus. These fibers were remote from the condensed chromatins, while F-actin in the cells undergoing apoptosis without CD accumulated around the nuclear fragments. The acceleration of AD-induced apoptosis by CD was found in U937 cells as well. Apoptosis induced by other DNA damaging agents (CDDP and MMC) was also accelerated by CD. The apoptosis by AD and CD was counteracted by antioxidants such as α-tocopherol and luteolin. Thus, the DNA damage-induced apoptosis is affected by disruption of the actin system, and probably, reactive oxygen species are involved in the process. 1 Exp. Hematol. , 27, Suppl. 1, 51 (1999).