Uncoordinated mutant number-45 myosin chaperone A (UNC-45A), a protein highly conserved throughout evolution, is ubiquitously expressed in somatic cells. It is correlated with tumorigenesis, proliferation, metastasis, and invasion of multiple malignant tumors. The current understanding of the role of UNC-45A in tumor progression is mainly related to the regulation of non-muscle myosin II (NM-II). However, many studies have suggested that the mechanisms by which UNC-45A is involved in tumor progression are far greater than those of NM-II regulation. UNC-45A can also promote tumor cell proliferation by regulating checkpoint kinase 1 (ChK1) phosphorylation or the transcriptional activity of nuclear receptors, and induces chemoresistance to paclitaxel in tumor cells by destabilizing microtubule activity. In this review, we discuss the recent advances illuminating the role of UNC-45A in tumor progression. We also put forward therapeutic strategies targeting UNC-45A, in the hope of paving the way the development of UNC-45A-targeted therapies for patients with malignant tumors.

Netrin-1, a secreted protein recently characterized as a relevant cancer therapeutic target, is the antiapoptotic ligand of the dependence receptors deleted in colorectal carcinoma and members of the UNC5H family. Netrin-1 is overexpressed in several aggressive cancers where it promotes cancer progression by inhibiting cell death induced by its receptors. Interference of its binding to its receptors has been shown, through the development of a monoclonal neutralizing antinetrin-1 antibody (currently in phase II of clinical trial), to actively induce apoptosis and tumor growth inhibition. The transcription factor p53 was shown to positively regulate netrin-1 gene expression. We show here that netrin-1 could be a target gene of the N-terminal p53 isoform Δ40p53, independent of full-length p53 activity. Using stable cell lines, harboring wild-type or null-p53, in which Δ40p53 expression could be finely tuned, we prove that Δ40p53 binds to and activates the netrin-1 promoter. In addition, we show that forcing immortalized human skeletal myoblasts to produce the Δ40p53 isoform, instead of full-length p53, leads to the up-regulation of netrin-1 and its receptor UNC5B and promotes cell survival. Indeed, we demonstrate that netrin-1 interference, in the presence of Δ40p53, triggers apoptosis in cancer and primary cells, leading to tumor growth inhibition in preclinical in vivo models. Finally, we show a positive correlation between netrin-1 and Δ40p53 gene expression in human melanoma and colorectal cancer biopsies. Hence, we propose that inhibition of netrin-1 binding to its receptors should be a promising therapeutic strategy in human tumors expressing high levels of Δ40p53.

Objective:To investigate the influence of short hairpin RNA (shRNA)mediated silencing YB - 1 on neuroblastoma SH - SY5Y cell proliferation and apoptosism then discuss regulatory mechanism. Methods:Establis-hing the small interfering RNA (siRNA)eukaryotic expression vector targeting YB - 1 of human neuroblastoma SH -SY5Y cells,transfection was performed using LipofectamineTM2000 liposome. The expression of YB - 1 protein was de-tected with Western blot after interference. The proliferation of SY5Y cells were detected by MTT assay. The apoptosis of cells was analyzed by flow cytometry. Detected the expression of CyclinD1,Bcl - 2 and Bax by Western blot. Re-sults:Expression of YB - 1 in transfected group (YB - 1 shRNA group)was lower than control group (SH - SY5Y group)(P < 0. 05). There was no significant difference between control group and invalid transfected group (NC group). MTT assay showed that cell proliferation level of YB - 1 shRNA group was significantly lower than SH -SY5 Y group. NC group was similar to SH - SY5 Y group. The number of apoptotic cells in YB - 1 shRNA group (23. 21 ± 1. 90)% was higher than SH - SY5 Y group (5. 05 ± 0. 83)% (P < 0. 01). There was no significant difference between NC group (6. 24 ± 1. 05)% and SH - SY5Y group (P > 0. 05)detected by flow cytometry analy-sis. The level of CyclinD1 and Bcl - 2 protein in YB - 1 shRNA group was lower than SH - SY5Y group. The level of Bax protein in YB - 1 shRNA group was more than SH - SY5Y group. NC group was similar to SH - SY5Y group that detected by Western blot. Conclusion:shRNA mediated silencing YB - 1 suppressed neuroblastoma SH - SY5Y cell to proliferate and promoted apoptosis by down regulating CyclinD1 and Bcl - 2,as well as up regulating Bax.

c-Kit is a classic proto-oncogene either mutated or upregulated in cancer cells, and this leads to its constitutive kinase activation and, thus, to uncontrolled proliferation. Although the pro-oncogenic role of c-Kit is of no doubt, some observations do not fit well with c-Kit solely as a tumor-promoting moiety. We show here that c-Kit actively triggers cell death in various cancer cell lines unless engaged by its ligand stem cell factor (SCF). This pro-death activity is enhanced when the kinase activation of c-Kit is silenced and is due to c-Kit intracellular cleavage by caspase-like protease at D816. Moreover, in vivo, overexpression of a c-Kit kinase-dead mutant inhibits tumor growth, and this intrinsic c-Kit tumor-suppressive activity is dependent on the D816 cleavage. Thus, c-Kit acts both as a proto-oncogene via its kinase activity and as a tumor suppressor via its dependence receptor activity.

Objective:To investigate the influence of shRNA - mediated silencing of Y - Box Binding Protein - 1 (YB - 1)on tumorigenesis of neuroblastoma cell SH - SY5Y in vivo. Methods:Establishing the small interfering RNA (siRNA)eukaryotic expression vector targeting YB - 1,transfection was performed using LipofectamineTM2000 liposomeinto human neuroblastoma SH - SY5Y cells. The expression of YB - 1 protein was detected with Western blot after interference. The effect of YB - 1 protein after inhibition on cell apoptosis was detected by plate clone formation assay. The effect of YB - 1 protein after inhibition on tumor growth in nude mice was estimated by measuring the size of tumor volume and weight. The effect of interferenceon the proliferation and apoptosis of tumor cells was assessed by HE staining and TUNEL analysis. Results:Compared with invalid transfection group (NC group),the expression of YB - 1 of control group (SH - SY5Y group)had no significant difference by Western blot,however,the expression level of YB - 1 of the transfection group (YB - 1 shRNA group)decreased significantly compared with the SH -SY5Y group,clone formation assay showed that colony formation ability of NC group cells compared with SH - SY5Y group cells did not change significantly (P > 0. 05),but YB - 1 shRNA group colony formation ability decreased sig-nificantly compared to the SH - SY5Y group (P < 0. 01). Statistical analysis of tumor growth in nude mice showed that the average tumor volume and average tumor weight of SH - SY5Y group was significantly higher than those of YB - 1 shRNA group (P < 0. 01),there was no difference about tumor volume and weight between NC group and SH- SY5Y group (P > 0. 05). HE staining and TUNEL analysis revealed that SH - SY5Y group and NC group com-pared with YB - 1 shRNA group had more active cell division ability and fewer apoptotic. Conclusion:shRNA - medi-ated silencing of YB - 1 led to a growth arrest and to induction of apoptosis,as well as significantly inhibited tumori-genesis of neuroblastoma cell in vivo.

INTRODUCTION:Bacterial infection and bile flow retardation form a vicious cycle which promotes stone formation and recurrence, and it seems that mucin overexpression plays an important role in this process. However, the mechanism of increased mucus secretion in the biliary tract by bacterial infection and its treatment remain unclear.MATERIAL AND METHODS:Human biliary epithelial cells were induced by neutrophil elastase (NE), and H2O2 production in the cell supernatants was detected by a specific kit, and then cells were pretreated with a H2O2 inhibitor, and expression of MUC5AC was detected by real-time polymerase chain reaction (PCR), Western blot, and immunohistochemistry. Moreover, selective PKC and Nox inhibitors, apocynin and bisindolylmaleimide I, were used to pretreat cells and detect H2O2, MUC5AC mRNA and protein expression. Then, we pretreated cells with selective inhibitors or NE, and detected transforming growth factor α (TGF-α) using an ELISA kit.RESULTS:H2O2 production increased in an NE dose-dependent manner (p < 0.001), and NE upregulated MUC5AC expression at both mRNA and protein levels, while DMTU, could reduce this high expression (p < 0.01 at mRNA level, p < 0.001 at grey analysis for western blot and p < 0.01 at mean density for immunohistochemical staining at protein level). Moreover, apocynin and bisindolylmaleimide I could reduce the H2O2 production stimulated by NE (p < 0.05), and reduce MUC5AC high expression (p < 0.01 at mRNA level, p < 0.001 at both grey analysis for western blot and mean density for immunohistochemical staining at protein level). In addition, NE induced TGF-α production, and any of the three selective inhibitors could reduce it (p < 0.05).CONCLUSIONS:NE-induced reactive oxygen species participated in the upregulation of MUC5AC production. Moreover, protein kinase C and NADPH oxidase (Nox) regulate MUC5AC production in NE-challenged human biliary epithelial cells.

Y-box binding protein-1 (YB-1), a member of Y-box protein family binding DNA and RNA, has been proposed as a novel marker in multiple malignant tumors and found to be associated with tumor malignancy. Neuroblastoma is an embryonal tumor arising from neuroblast cells of the autonomic nervous system, which is the most common cancer diagnosed in infants. It has been reported that YB-1 is highly expressing in various human tumors including nasopharynx, thyroid, lung, breast, colon, ovary, and prostate cancers. This study aimed to investigate the functional role of YB-1 in neuroblastoma by silencing YB-1 using RNA interference (shRNA) in neuroblastoma SH-SY5Y cells. We found that silencing of YB-1 decreased the proliferation, migration, and invasion of SH-SY5Y cells. At molecular level, inhibition of YB-1 decreased the expression level of PCNA as well as MMP-2 in neuroblastoma SH-SY5Y cells. Also, we discovered that YB-1 silencing sensitized SH-SY5Y cells to cisplatin and promoted the apoptosis induced by cisplatin due to down-regulation of multidrug resistance (MDR) 1 protein via NF-κB signaling pathway. Therefore, we consider that targeting YB-1 is promising for neuroblastoma treatment and for overcoming its cisplatin resistance in the development of new neuroblastoma therapeutic strategies.

Y-box binding protein-1 (YB-1), a member of cold-shock protein superfamily, has been demonstrated to be associated with tumor malignancy, and is proposed as a prognostic marker in multiple carcinomas. However, the role of YB-1 in neuroblastoma has not been well studied. To investigate the functional role of YB-1 in neuroblastoma, we established a YB-1-silenced neuroblastoma cell strain by inhibiting YB-1 expression using a shRNA knockdown approach. YB-1-silenced neuroblastoma SH-SY5Y cells exhibited a pronounced reduction in cell proliferation and an increased rate of apoptosis in vitro and in vivo xenograft tumor model. At molecular level, YB-1 silencing resulted in downregulation of Cyclin A, Cyclin D1 and Bcl-2, as well as upregulated levels of Bax, cleaved caspase-3 and cleaved PARP-1. We further demonstrated that YB-1 transcriptionally regulated Cyclin D1 expression by chromatin-immunoprecipitation and luciferase reporter assays. In addition, xenograft tumors derived from neuroblastoma SH-SY5Y cell line were treated with YB-1 shRNA plasmids by intra-tumor injection, and YB-1 targeting effectively inhibited tumor growth and induced cell death. In summary, our findings suggest that YB-1 plays a critical role in neuroblastoma development, and it may serve as a potential target for neuroblastoma therapy.

UNC5H4 is a newly identified member of the UNC5H receptor family. Previously, we have demonstrated that UNC5H4 expression is significantly higher in favorable neuroblastomas than in unfavorable ones, and higher UNC5H4 level is correlated with longer survival time. However, the function of UNC5H4 in the tumorigenesis of neuroblastoma still remains elusive. In the present study, the effects of UNC5H4 overexpression on neuroblastoma SH-SY5Y cells were investigated. We showed that enforced expression of UNC5H4 receptor significantly inhibited anchorage-dependent and anchorage-independent growth of SH-SY5Y cells. Cell migration and invasion of SH-SY5Y cells transfected with UNC5H4-expressing plasmid were obviously suppressed as compared to those transfected with emptor vector or non-transfected cells. Moreover, overexpression of UNC5H4 resulted in apoptosis in SH-SY5Y cells. The induction of apoptosis by UNC5H4 was completely abolished in the presence of its ligand, netrin-1. Finally, caspase cleavage and the presence of death domain were required for UNC5H4 to induce apoptosis in neuroblastoma SH-SY5Y cells. These data suggest that the dependence receptor UNC5H4 may act as a putative tumor suppressor in neuroblastoma.

The mechanism of apoptosis via the p53‑dependent pathway remains to be fully understood. In the present study, a novel p53 target gene, Unc5D, was identified and its possible function in human neuroblastoma cells was investigated. The apoptotic effects of Unc5D in SK‑N‑BE (p53‑/‑) and SH‑SY5Y (p53+/+) cells were measured by an 3‑(4,5‑dimethylthiazol‑2‑yl)2,5‑diphenyltetrazolium bromide solution assay. Reverse transcription‑polymerase chain reaction (RT‑PCR) was also performed to detect the endogenous expression of Unc5D. In H1299 (p53‑/‑) cells, following overexpression of p53, RT‑PCR and western blot analysis were used to detect the Unc5D mRNA and protein levels. In order to detect the promoter activity in the Unc5D gene, a luciferase assay was performed. Finally, to confirm the activate site of p53 subsequent to DNA damage, western blot analysis was used to analyze the phosphorylation site of Unc5D stable and mock clones in H1299 cells by co‑expression of p53. Unc5D‑induced apoptosis may be largely dependent on the p53 status. Notably, Unc5D was found to be a direct transcriptional target of p53. During adriamycin‑mediated apoptosis, Unc5D was significantly induced in p53‑proficient SH‑SY5Y cells but not in p53‑deficient SK‑N‑BE cells. Overexpression of p53 resulted in an increase in the expression levels of endogenous Unc5D. Additionally, two elements were identified in the sequence of Unc5D. Notably, Unc5D expression also induced phosphorylation of p53 at serine‑15. Unc5D is thus a newly identified transcriptional target of pro‑apoptotic p53 and may also act upstream of p53 to induce p53‑dependent apoptosis by phosphorylation at ser‑15.

UNC5H4 is a netrin-1 receptor UNC5H family member. In this study, we found that UNC5H4 is a direct transcriptional target of p53. During adriamycin (ADR)-mediated apoptosis, UNC5H4 was significantly induced in p53-proficient U2OS cells but not in p53-deficient H1299 cells. Enforced expression of p53 induced UNC5H4. Consistent with these results, siRNA-mediated knockdown of p53 in U2OS cells attenuated ADR-dependent induction of UNC5H4. Indeed, we found four putative p53-responsive elements within intron 1 of UNC5H4 gene. Luciferase reporter assay and ChIP analysis demonstrated that, among them, two tandem elements respond to exogenous p53 which is efficiently recruited onto them. Furthermore, enforced expression of UNC5H4 remarkably reduced number of drug-resistant colonies in p53-proficient cells but not in p53-deficient cells, suggesting that UNC5H4-induced apoptosis is dependent on p53 status. siRNA-mediated knockdown of UNC5H4 rendered U2OS cells resistant to ADR. Collectively, our present results suggest that UNC5H4 amplifies p53-dependent apoptotic response.

Aquaporins (AQP) are integral membrane proteins that serve as channels in the transfer of water, and in some cases, small solutes across the membrane. In order to detect the expression of AQP1～10 mRNA in the human pleural mesothelial cells, culture reproducible model of human pleural mesothelial cells was established in vitro.The value of aquaporin water channels was investigated in pleural fluid dynamics. Mesothelial cells were isolated from pleural effusion fluid of nonmalignant pleural effusion patients and cultured in vitro. The mesothelial cells were identified by morphology and streptomyces protein-peroxidase(S-P) procedure. RT-PCR was used to detect the expression of AQP1～10 mRNA in human pleural mesothelial cells. Establishment of culture reproducible model achieved success of human pleural mesothelial cells in vitro. Immunocytochemistry revealed that cytokeratin and vimentin expressed positive, VⅢ factor associated antigen and CD45 was negtive. Confluent human pleural mesothelial cells appeared multipolar and like cabblestone.Under electron microscopy numerous surface microvilli and abundant endoplasmic reticulum were observed. The cells was identified that it was mesothelial cells. The cells isolated from human pleural effusion fluid have higher purity quotient . RT-PCR studies showed that all of the AQP1～10 mRNA expressed in mesothelial cells. AQP1, AQP9, AQP10 mRNA have abundant expression in mesothelial cells and significant deviation was found between AQP1, AQP9, AQP10 and AQP2, AQP3, AQP7, AQP8. Human pleural mesothelial cells expressed AQP1～10 mRNA, all of them have relation with the function of human pleural mesothelial cells. It was confirmed that mesothelial cells have important contribution in pleural fluid dynamics. Aquaporins (AQP) are involved in rapid and active gating of water across biological membranes.Expression of aquaporins was intervened, new treat method may be found of pleural effusion.

Le proto-oncogène c-Kit inhibe la croissance tumorale en agissant comme un récepteur à dépendance C-Kit est généralement considéré comme un récepteur à tyrosine kinase et comme un proto-oncogène, dont la surexpression et la mutation conduisent à une progression tumorale médiée par son activité kinase. En clinique, les traitements ciblant l’activité kinase de c-Kit, comme l’Imatinib (Gleevec), ont été largement utilisés pour traiter les patients atteints de maladies liées à c-Kit. Alors que le rôle de c-Kit comme proto-oncogène ne fait aucun doute, certaines études et analyses de bases de données diffèrent avec l’idée d’un rôle pro-tumoral de c-Kit, laissant penser à un rôle différent de c-Kit dans le cancer. Ici, nous montrons que c-Kit appartient à la famille des récepteurs à dépendance, de la même façon que d’autres récepteurs de la famille des tyrosine kinases tel que MET, RET et TrkC. En absence de son ligand Stem Cell Factor (SCF), au lieu de rester inactif, c-Kit déclenche l’apoptose, qui peut être renforcée par l’invalidation de son activité kinase. En parallèle, nous montrons que c-Kit est capable de se lier à la caspase-9 et de l’activer. De plus, à la manière d’autres récepteurs à dépendance, c-Kit est aussi clivé par des protéases de type caspase sur son résidu acide aspartique D816, qui est nécessaire à son activité pro-apoptotique. La mutation du site D816 inhibe l’interaction entre c-kit et la caspase-9 et invalide l’activité pro-apoptotique de c-Kit. De façon intéressante, la mutation D816 est l’une des mutations les plus communes de ce récepteur dans la plupart des cancers liés à c-Kit, et cette mutation favorise la résistance au traitement Gleevec. Nous montrons aussi que la surexpression de c-Kit invalidé pour son activité kinase est capable d’inhiber la croissance tumorale dans des modèles animaux, alors que la mutation du site D816 empêche son effet suppresseur de tumeur. En outre, nous avons développé un outil permettant de bloquer l’interaction entre SCF et c-Kit, déclenchant l’activité pro-apoptotique de c-Kit dans les cancers positifs pour ce récepteur. En utilisant l’activité pro-apoptotique de c-Kit, en combinaison avec des inhibiteurs de kinases comme le Gleevec, nous proposons une nouvelle stratégie thérapeutique. En conclusion, nous démontrons que c-Kit est un membre de la famille des récepteurs à dépendance, présentant une activité pro-apoptotique, et pouvant être utilisé comme un outil alternatif dans le cadre d’un traitement contre le cancer