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Background Uveal melanoma (UM) is a severe human malignancy with a high mortality rate, as well as high metastasis and recurrence potential. The active mutation of G protein subunit alpha q (GNAQ) or G protein subunit alpha 11 (GNA11) is a major trigger for UM. Oncolytic adenovirus H101 (H101) is the first oncolytic virus approved for clinical applications in cancer therapy by the China Food and Drug Administration. We investigated whether combining H101 with the downregulation of GNAQ expression would act synergistically in UM therapy. Methods Three UM cell lines OMM2.3 and 92.1, harboring GNAQ mutation, and OCM1, harboring B-Raf proto-oncogene mutation, were chosen for our research. The cellular toxicity of adenoviral infection and the cell growth rate were measured with a Cell Counting Kit-8. Western blot analysis was used to detect GNAQ, p-MEK1/2, YAP, and p-YAP expression. The apoptosis and cell-cycle distribution of cells were evaluated with annexin-V and propidium iodide staining. Results Our results revealed that OMM2.3 and 92.1 cells were more sensitive to H101 infection than OCM1 cells. GNAQ expression was markedly reduced by small interfering RNA, siGNAQ. Combined treatment of siGNAQ and H101 inhibited the proliferation and activated the apoptosis of OMM2.3 and 92.1 cells by blocking the phosphorylation of MEK1/2 and increasing the phosphorylation of YAP. Conclusions In summary, a therapy combining H101 and siGNAQ is feasible, with potential utility as a novel targeted molecular therapy for UM, especially those carrying a GNAQ mutation.

Uveal melanoma (UM) is an intraocular malignant tumor with a high mortality rate. Recent studies have shown the functions of long non-coding RNAs (lncRNAs) in tumorigenesis; thus, targeting tumor-specific lncRNA abnormalities has become an attractive approach for developing therapeutics to treat uveal melanoma. In this study, we identified a novel nuclear CANT1 lncRNA (CASC15-New-Transcript 1) that acts as a necessary UM suppressor. CANT1 significantly reduced tumor metastatic capacity and tumor formation, either in cell culture or in animals harboring tumor xenograft. Intriguingly, XIST lncRNA serves as a potential target of CANT1, and JPX or FTX lncRNA subsequently serves as a contextual hinge to activate a novel CANT1-JPX/FTX-XIST long non-coding (lncing) pathway in UM. Moreover, CANT1 triggers the expression of JPX and FTX by directly binding to their promoters and promoting H3K4 methylation. These observations delineate a novel lncing cascade in which lncRNAs directly build a lncing cascade without coding genes that aims to modulate UM tumorigenesis, thereby specifying a novel "lncing-cascade renewal" anti-tumor therapeutic strategy by correcting aberrant lncing cascade in uveal melanoma.

Uveal melanoma (UM) is the most frequently occurring primary intraocular malignancy in adults. Tyrosinase (TYR) is a copper-containing enzyme and a type I membrane protein that is involved in the generation of melanin, the main pigment in vertebrates. TYR-related protein 1 (TYRP1) is regarded to have a crucial role in the immunotherapy of melanoma. As biomarkers, the TYR-related proteins, TYRP1 and TYRP2, exhibit specific expression in melanocytes, while also contributing to melanin synthesis within melanosomes. In the present study, the differential expression of TYRP1 was investigated at the mRNA, protein and morphological levels in four human UM cell lines (SP6.5, OM431, OCM1 and OCM290) and the human retinal pigment epithelium (RPE) cell line, using polymerase chain reaction, western blotting, immunocytochemistry and immunofluorescence staining. It was found that SP6.5 cells expressed the highest level of TYRP1, in comparison to SP6.5 OCM1 and OM431 cells, which produced less TYRP1, and OCM290 cells, which produced almost no TYRP1. No TYRP1 protein expression was identified in the RPE cell line. These findings indicate the potential use of TYRP1 in the development of therapy for UM.

A long noncoding RNA directly builds an intrachromosomal interaction complex to establish allele-specific transcriptional gene silencing over a large chromosomal domain., Kcnq1ot1 is a long noncoding ribonucleic acid (RNA; lncRNA) that participates in the regulation of genes within the Kcnq1 imprinting domain. Using a novel RNA-guided chromatin conformation capture method, we demonstrate that the 5′ region of Kcnq1ot1 RNA orchestrates a long-range intrachromosomal loop between KvDMR1 and the Kcnq1 promoter that is required for maintenance of imprinting. PRC2 (polycomb repressive complex 2), which participates in the allelic repression of Kcnq1, is also recruited by Kcnq1ot1 RNA via EZH2. Targeted suppression of Kcnq1ot1 lncRNA prevents the creation of this long-range intrachromosomal loop and causes loss of Kcnq1 imprinting. These observations delineate a novel mechanism by which an lncRNA directly builds an intrachromosomal interaction complex to establish allele-specific transcriptional gene silencing over a large chromosomal domain.

Aim: Hypoxia, a characteristic of almost all types of solid tumors, has been associated with poor outcome in a number of human malignancies. The aim of this study was to investigate the molecular mechanisms involved in hypoxia-induced activation of the human survivin gene promoter in cervical HeLa cells. Material and Methods: Immunohistochemical staining was used to detect the expression of HIF-1α and survivin in cervical cancer samples and normal cervical samples. Under normoxic and hypoxic conditions, the expression of hypoxia inducible factor (HIF)-1α and survivin in cervical cancer HeLa cells was detected by quantitative reverse transcription polymerase chain reaction and Western blotting. Luciferase reporter assays was used to investigate the molecular mechanisms in hypoxia-induced survivin activation. We also studied the effect of HIF-1α overexpression on the expression of survivin in cervical cancer HeLa cells. Results: Significant HIF-1α and survivin overexpression is associated with cervical cancer, and HIF-1α protein expression is strongly correlated with survivin protein expression. In cervical cancer cell line (HeLa), hypoxia upregulated both HIF-1α and survivin expression. Moreover, luciferase reporter assays using survivin core promoter demonstrated that survivin transcription was activated under hypoxia conditions and was associated with HIF-1α overexpression. The transcriptional activation of reporter genes in response to hypoxia is independent of potential HIF-1α-responsive element, located between −86 and −82 regions. HIF-1α overexpression significantly activated survivin expression. Conclusion: Our results demonstrate that survivin expression is upregulated following the induction of HIF-1α by hypoxia resulting from tumor formation, possibly leading to tumor progression. These findings have potential implication in developing novel cancer therapy targeting HIF-1.

Vitamin K2 (VK2) exerts cell growth inhibitory effects in various human cancer cells such as SMMC-7721 hepatocellular carcinoma (HCC) cells. BCL-2 is an antiapoptotic protein that is frequently overexpressed in numerous tumors. Modulation of multiple antiapoptotic signaling pathways involving BCL-2, which are related to growth factor-stimulated signal transduction in cell survival, is essential for enhancement of the cytotoxic effect of anticancer drugs. In this study, we tested a new strategy of gene therapy by combining BCL-2 siRNA with VK2. In SMMC-7721 HCC cells, the combined treatment significantly enhanced cytotoxicity compared with treatment with either VK2 or siBCL-2 alone. We found that combined treatment induced a significantly different level of G2 stage inhibition. Furthermore, the p53 protein was overexpressed 24 h subsequent to combination treatment, and p21 was clearly increased at 36 h as a consequence of the increased p53 activity. In conclusion, these data suggest that the antitumor effect of VK2 may be improved by silencing BCL-2 expression in SMMC-7721 HCC cells and provides support for the combined use of VK2 and siBCL-2 as a promising approach in cancer gene therapy.

Targeting tumor-specific gene abnormalities has become an attractive approach in developing therapeutics to treat cancer. Overexpression of Bcl2 and mutations of p53 represent two of the most common molecular defects in tumors. In the nucleus, p53 induces cell cycle arrest, while it interacts with Bcl2 outside of the nucleus to regulate signal pathways involved in apoptosis. To potentiate antitumor activity, we tested a "double target" approach to antitumor therapy by combining H101, a recombinant oncolytic adenovirus that targets the inactive p53 in tumors, with a small interfering RNA (siBCL2) that targets Bcl2. In cell culture, the combined treatment significantly enhanced apoptosis and cytotoxicity as compared with treatment with either H101 or siBCL2 alone. In animals carrying tumor xenographs, combined H101 and siBCL2 treatment significantly inhibited tumor growth and prolonged survival. At the end of the study, all animals in the combined therapy group survived and two of the five animals showed complete eradication of their tumors. Interestingly, siBCL2 treatment increased H101 viral replication in both treated cells and tumor tissues. Simultaneously targeting two tumor-specific gene abnormalities using an oncolytic adenovirus and siRNA potentiates total antitumor activity.

Survivin, a unique member of the inhibitor of apoptosis protein family, is overexpressed in many cancers and considered to play an important role in oncogenesis. In this study, we cloned and identified the proximal 269 bp promoter of survivin gene, which exhibited strong promoter activity in HeLa cells. The TATA-less, GC-rich promoter contains 7 putative binding sites for Sp1, two of which (one at position −148 to −153, the other at position −127 to −140) are essential in regulating basal survivin promoter activity. Not only Sp1 but also Sp3 can activate the survivin promoter, which were proven by EMSA, blocking Sp1 or Sp3 using RNAi or mithramycin treatment of HeLa cells, and overexpression of Sp1 or Sp3. Our results collectively suggest that Sp1 cooperates with Sp3 to regulate survivin promoter activity.

The Notch proteins constitute a family of transmembrane receptors that play a pivotal role in cellular differentiation, proliferation, and apoptosis. RNA interference of Presenilin1 (PS1) and Notch1 was carried out in this research to determine whether it could block Notch signaling and induce growth inhibition in HeLa cells. We transfected synthesized target small interfering RNA (siRNA) into HeLa cells, and blocking of Notch signaling was detected by C-promoter binding factor-1 (CBF1) reporter. We then conducted cell proliferation assay. Cells transfected with PS1 and Notch1 siRNA showed great inhibition in proliferation compared to the controlsin vitroandin vivo. We conclude that RNA interference of PS1 or Notch1 can block Notch signaling and consequently induce growth inhibition of HeLa cells.

After the publication of this work [1], we noticed an error whereby two corresponding author’s names were missed from the Acknowledgements section. The corrected statement is provided below

Background Long non-coding RNAs (lncRNAs) are not translated into proteins and were initially considered to be part of the ‘dark matter’ of the genome. Recently, it has been shown that lncRNAs play a role in the recruitment of chromatin modifying complexes and can influence gene expression. However, it is unknown if lncRNAs function in a similar way in cancer. Results Here, we show that the lncRNA ROR occupies and activates the TESC promoter by repelling the histone G9A methyltransferase and promoting the release of histone H3K9 methylation. Suppression of ROR in tumors results in silencing of TESC expression, and G9A-mediated histone H3K9 methylation in the TESC promoter is restored, which significantly reduces tumor growth and metastasis. Without ROR silencing, TESC knockdown presents consistent and significant reductions in tumor progression. Conclusions Our results reveal a novel mechanism by which ROR may serve as a decoy oncoRNA that blocks binding surfaces, preventing the recruitment of histone modifying enzymes, thereby specifying a new pattern of histone modifications that promote tumorigenesis. Electronic supplementary material The online version of this article (doi:10.1186/s13059-015-0705-2) contains supplementary material, which is available to authorized users.

The insulin-like growth factor II (IGF2) gene is aberrantly expressed in tumors as a result of loss of imprinting (LOI). Reactivation of the normally-suppressed maternal allele may lead to IGF2 upregulation and increased tumor growth, particularly in colon cancer. However, the mechanisms underlying IGF2 LOI in tumors are poorly defined. In this report, we identified polycomb repressive complex 2 (PRC2) docking factor SUZ12 as a critical factor in regulating IGF2 imprinting in tumors. Human colon cancer cell lines (HRT18 and HT29) show loss of IGF2 imprinting. Ectopic expression of SUZ12 restored normal monoallelic expression of IGF2 in these two colon cancer cell lines. Using chromatin immunoprecipitation (ChIP) and chromatin conformation capture (3C), we found that the virally-expressed SUZ12 bound to IGF2 promoters, coordinating with endogenous CTCF to orchestrate a long range intrachromosomal loop between the imprinting control region (ICR) and the IGF2 promoters. The histone methyltransferase EZH2 was recruited to the IGF2 promoters, where it induced H3K27 hypermethylation, suppressing one allele, leading to the restoration of IGF2 imprinting. These data demonstrate that SUZ12 is a key molecule in the regulation of monoallelic expression of IGF2, suggesting a novel epigenetic therapeutic strategy for modulating IGF2 production in human tumors.

MARs are not only the structure bases of chromatin higher order structure but also have much biological significance. In this study, the whole sequence of about 100 kb in length from BAC clone of GS1-223D4 (GI: 5931478), in which human PI3Kgamma gene is localized, was analyzed by two online-based computer programs, MARFinder and SMARTest. A strong potential MAR was predicted in the last and largest intron of PI3Kgamma. The predicted 2 kb MAR, we refer to PIMAR, was further analyzed through biochemical methods in vitro and in vivo. The results showed that the PIMAR could be associated with nuclear matrices from HeLa cells both in vitro and in vivo. Further reporter gene analysis showed that in the transient transfection the expression of reporter gene linked with reversed PIMAR was repressed slightly, while in stably integrated state, the luciferase reporter both linked with reversed and orientated PIMAR was enhanced greatly in NIH-3T3 and K-562. These results suggest that the PIMAR maybe has the capacity of shielding integrated heterogeneous gene from chromatin position effect. Through combination of computer program analysis with confirmation by biochemical methods, we identified, for the first time, a 2 kb matrix attachment region like sequence in the last intron of human PI3Kgamma.

The Hedgehog (Hh) signaling pathway has been demonstrated to play a critical role in controlling embryonic development, tissue patterning, wound healing and a variety of cell functions. Aberrant activation of Hh signaling is implicated in the pathogenesis of many human cancers, and in angiogenesis. However, the role of this pathway in uveal melanoma (UM) carcinogenesis remains unknown. In this study, we investigated the effects of Hh inhibition using the specific Smoothened (Smo) antagonist cyclopamine to block Hh signaling in cultured human UM cell lines expressing Hh signaling components. Cyclopamine treatment effectively increased apoptosis and inhibited cell proliferation, migration, and epithelial-to-mesenchymal transition (EMT) by downregulating the Hh final arbiter glioblastoma 1 (Gli1), which regulates the transcription of target genes in the nucleus. Changes in gene and protein expression levels were detected by real-time PCR and by western blotting and immunocytochemistry, respectively. Cell cycle and apoptosis regulation induced by cyclopamine were demonstrated by flow cytometry. In addition, the migration capability of UM cells was reduced, as demonstrated by transwell migration and scratch assays. The effects of Hh inhibition on the levels of angiogenesis factors secreted by UM cells were examined by tube-formation assay. Conclusion: Blocking the Hh pathway by cyclopamine decreased cell viability, migration, EMT, and angiogenesis, increased apoptosis, and induced G1 phase cell cycle arrest in UM cells. Collectively, these results provide the first evidence of the significance of Gli1 activation downstream of Smo as a therapeutic target and the potential value of cyclopamine for the treatment of human UM.

Apoptosis and autophagy are crucial mechanisms regulating cell death, and the relationship between apoptosis and autophagy in the liver has yet to be thoroughly explored. TIGAR (TP53-induced glycolysis and apoptosis regulator), which is a p53-inducible gene, functions in the suppression of ROS (reactive oxygen species) and protects U2OS cells from undergoing cell death. In this study, silencing TIGAR by RNAi (RNA interference) in HepG2 cells down-regulated both TIGAR mRNA (∼75%) and protein levels (∼80%) and led to the inhibition of cell growth (P

Hypoxia, a characteristic of almost all types of solid tumors, has been associated with poor outcome in a number of human malignancies. The aim of this study was to investigate the molecular mechanisms involved in hypoxia-induced activation of the human survivin gene promoter in cervical HeLa cells.Immunohistochemical staining was used to detect the expression of HIF-1α and survivin in cervical cancer samples and normal cervical samples. Under normoxic and hypoxic conditions, the expression of hypoxia inducible factor (HIF)-1α and survivin in cervical cancer HeLa cells was detected by quantitative reverse transcription polymerase chain reaction and Western blotting. Luciferase reporter assays was used to investigate the molecular mechanisms in hypoxia-induced survivin activation. We also studied the effect of HIF-1α overexpression on the expression of survivin in cervical cancer HeLa cells.Significant HIF-1α and survivin overexpression is associated with cervical cancer, and HIF-1α protein expression is strongly correlated with survivin protein expression. In cervical cancer cell line (HeLa), hypoxia upregulated both HIF-1α and survivin expression. Moreover, luciferase reporter assays using survivin core promoter demonstrated that survivin transcription was activated under hypoxia conditions and was associated with HIF-1α overexpression. The transcriptional activation of reporter genes in response to hypoxia is independent of potential HIF-1α-responsive element, located between -86 and -82 regions. HIF-1α overexpression significantly activated survivin expression.Our results demonstrate that survivin expression is upregulated following the induction of HIF-1α by hypoxia resulting from tumor formation, possibly leading to tumor progression. These findings have potential implication in developing novel cancer therapy targeting HIF-1.

Treatment trends of retinoblastoma (RB) have gradually evolved from eye enucleation and external radiation to local treatment. Combined treatment with an oncolytic virus and chemotherapy is currently a new method in RB treatment. To investigate the therapeutic effect of oncolytic adenovirus SG600 in combination with vincristine (VCR) on retinoblastoma in vitro, the cell viability, cell cycle effects and apoptotic activity of HXO-RB44 cells treated with SG600, VCR or SG600 plus VCR were measured using a cell counting kit-8-based procedure and flow cytometry. Western blot analysis for Akt, p-Akt, p-p53 and p-Rb protein was performed to investigate the underlying mechanisms of combined therapy. The combination therapy exerted a synergistic antitumor effect via a type of G2/M and S phase arrest rather than the induction of apoptosis. The combination of VCR and SG600 further reduced Akt phosphorylation compared with cells treated with VCR alone, suggesting that SG600 could overcome chemoresistance, perhaps by down-regulating Akt in RB cells. An increase in the expression of p-p53 and decrease in p-Rb expression in HXO-RB44 after co-treatment might be associated with cell cycle block. Western blot examination revealed that VCR might enhance SG600 replication. These results suggest that viro-chemo combination therapy is a feasible and potentially promising approach for the treatment of retinoblastoma.

There is a need for more effective treatments for uveal melanoma. The recombinant oncolytic adenovirus H101 replicates specifically in p53-depleted tumor cells, and has been approved for use by the Chinese State Food and Drug Administration. However, this treatment is associated with subsequent remission. Transfection of uveal melanoma cells with a small interfering RNA against Notch1 (siNotch1) effectively suppressed Notch1 expression, resulting in significant cell growth inhibition when combined with H101 treatment. Combined treatment with siNotch1 and H101 (H101-Notch1-siRNA) greatly enhanced apoptosis and cell cycle arrest in vitro as compared to treatment with H101 or siNotch1 alone. For in vivo treatments, the combined treatment of siNotch1 and H101 showed remarkable tumor growth inhibition and prolonged mouse survival in the OCM1 xenograft model. We predict that Notch pathway deregulation could be a feature of uveal melanoma, and could be a therapeutic target, especially if p53 is concurrently targeted.

The human WWOX gene, known as WW domain-containing oxidoreductase, is located on 16q23.3-24.1, a chromosome region that spans the common fragile site, FRA16D. Abnormal transcripts or even loss of expression are frequently found in a number of cancer cell types, including breast, ovarian, prostate and lung cancer cells. It has therefore been proposed that the WWOX gene encodes a candidate tumor suppressor, possibly a pro-apoptotic protein. However, the mechanism behind this is not entirely clear. In the present study, we examined the pro-apoptotic action of WWOX using transient expression in A549 cells. We observed that the ectopic expression of WWOX caused apoptosis in A549 cells. We further observed procaspase-3 and procaspase-9 activation and the release of cytochrome C from the mitochondria in A549 cells transfected with pcDNA3.0-WWOX. These data indicate that WWOX induces apoptosis in A549 cells via the mitochondrial pathway.

Hypoxia is an essential feature of retinoblastoma and contributes to poor prognosis and resistance to conventional therapy. MicroRNAs (miRNAs) are small non-coding RNAs involved in a wide variety of biological processes, including cell differentiation, proliferation, death and metabolism. However, the relationship between hypoxia and the expression of miRNAs in retinoblastoma is not well understood. In this study, we aimed to analyze the pattern of miRNA expression in a retinoblastoma cell line under hypoxic conditions and to identify the miRNAs regulated by hypoxia, as well as their possible functions. miRNA expression profiling in retinoblastoma cells (HXO-RB44) under normal and hypoxic conditions was assessed by microarray techniques. The differentially expressed miRNAs were subjected to bioinformatic analyses to predict and categorise the key miRNAs and their target genes. A quantitative real-time RT-PCR approach was used to validate their expression. A Cell Counting kit was used to evaluate the functional significance of miR-181b in RB cell proliferation. There were 46 miRNAs that changed expression more than 2-fold in response to hypoxia (34 up-regulated and 12 down-regulated). We identified a cluster of miRNAs that includes miR-181b, miR-125a-3p, miR-30c-2, miR-497 and miR-491-3p as hypoxia-regulated miRNAs (HRMs) in retinoblastoma cells, of which miR-181b was the most typically differentially expressed miRNA under hypoxic conditions. Functionally, these HRMs are involved in apoptosis, cell adhesion, cell proliferation and mRNA processing, all processes that associate closely with the hypoxia response of cancer cells. Additionally, we found that administration of miR-181b inhibitor can suppress proliferation of retinoblastoma cells. These findings provide the first evidence that miRNAs play an important role in the hypoxia response of retinoblastoma cells. MiR-181b, the most typically up-regulated miRNA may aid in future clinical intervention of retinoblastoma.

There is a growing evidence that regucalcin (RGN) plays a multifunctional role in liver cancer cells. Previous reports showed that the presence of phorbol 12-myristate 13-acetate (PMA) caused a significant increase in RGN mRNA expression and promoter activity in rat hepatoma cells. In this study, we confirmed that human RGN is also up-regulated by PMA treatment independent of translation, and we identified the mechanism by which PMA up-regulates the expression of human RGN via driving SP1 away from a SP1 motif located within −188/−180 of the promoter in HepG2 cells. Overexpression of SP1 dramatically reduces PMA-induced up-regulation of both internal expression of mRNA and promoter activity, whereas knockdown of SP1 has the opposite effect. Therefore, the present study delineates the fundamental elements in the promoter which will be helpful in the future studies on the regulation of RGN expression in liver cancer.

Tumor suppressor microRNA miR-145 is commonly down-regulated in colon carcinoma tissues, but its specific role in tumors remains unknown.In this study, the authors identified the Friend leukemia virus integration 1 gene (FLI1) as a novel target of miR-145. FLI1 is involved in t(11;22)(q24:q12) reciprocal chromosomal translocation in Ewing sarcoma, and its expression appears to be associated with biologically more aggressive tumors.The authors demonstrated that miR-145 targets a putative microRNA regulatory element in the 3'-untranslated region (UTR) of FLI1, and its abundance is reversely associated with FLI1 expression in colon cancer tissues and cell lines. By using a luciferase/FLI1 3'-UTR reporter system, they found that miR-145 down-regulated the reporter activity, and this down-regulation was reversed by anti-miR-145. Mutation of the miR-145 microRNA regulatory element sequence in the FLI1 3'-UTR abolished the activity of miR-145. miR-145 decreased FLI1 protein but not FLI1 mRNA, suggesting a mechanism of translational regulation. Furthermore, the authors demonstrated that miR-145 inhibited cell proliferation and sensitized LS174T cells to 5-fluorouracil-induced apoptosis.Taken together, these results suggest that miR-145 functions as a tumor suppressor by down-regulating oncogenic FLI1 in colon cancer.

Background MicroRNA-145 (miR-145) is considered to play key roles in many cellular processes, such as proliferation, differentiation and apoptosis, by inhibiting target gene expression. DNA Fragmentation Factor-45 (DFF45) has been found to be the substrate of Caspase-3, and the cleavage of DFF45 by caspase-3 during apoptosis releases DFF40 that degrades chromosomal DNA into nucleosomal fragments. There are currently no in-depth studies on the relationship between miR-145 and the DFF45 gene. Results In this study, we identified DFF45 as a novel target of miR-145. We demonstrated that miR-145 targets a putative binding site in the coding sequence (CDS) of DFF45, and its abundance is inversely associated with DFF45 expression in colon cancer cells. Using a luciferase reporter system, we found that miR-145 suppresses the expression of the luciferase reporter gene fused to the putative binding site of DFF45. The level of DFF45 protein, but not DFF45 mRNA, was decreased by miR-145, suggesting a mechanism of translational regulation. Furthermore, we demonstrate that this specific silencing of DFF45 by miR-145 accounts, at least in part, for the staurosporine-induced tumor cell apoptosis in vitro. Conclusions Our study reveals a previously unrecognized function of miR-145 in DFF45 processing, which may underlie crucial aspects of cancer biology.

Nox4, a member of Nox family of NADPH oxidase expressed in nonphagocytic cells, is a major source of reactive oxygen species in many cell types. But understanding of the role of Nox4 in the production of ROS and of regulation mechanism of oxidase activity is largely unknown. This study reports for the first time the generation and characterization of 5 mAbs against a recombinant Nox4 protein (AA: 206-578). Among 5 novel mAbs, 3 mAbs (8E9, 5F9, 6B11) specifically recognized Nox4 protein in HEK293 transfected cells or human kidney cortex by western blot analysis; mAb 8E9 reacted with intact tet-induced T-REx™ Nox4 cells in FACS studies. The other 2 mAbs 10B4 and 7C9 were shown to have a very weak reactivity after purification. Immunofluorescence confocal microscopy showed that Nox4 localized not only in the perinuclear and endoplasmic reticulum regions but also at the plasma membrane of the cells which was further confirmed by TIRF-microscopy. Epitope determination showed that mAb 8E9 recognizes a region on the last extracellular loop of Nox4, while mAbs 6B11 and 5F9 are directed to its cytosolic tail. Contrary to mAb 6B11, mAb 5F9 failed to detect Nox4 at the plasma membrane. Cell-free oxidase assays demonstrated a moderate but significant inhibition of constitutive Nox4 activity by mAbs 5F9 and 6B11. In conclusion, 5 mAbs raised against Nox4 were generated for the first time. 3 of them will provide powerful tools for a structure/function relationship of Nox4 and for physiopathological investigations in humans.

The p53 tumor suppressor pathway is impaired in more than 90% of cervical cancers and cancer-derived cell lines as a result of infection by human papillomavirus (HPV). The HPV E6 oncoprotein forms complexes with p53 and promotes its degradation via the ubiquitin-dependent mechanism. In this study, we attempted to improve the clinical outcomes of this combined therapy by modifying the p53-targeted adenovirus to become radiation-responsive. The anti-tumor adenovirus was constructed by inserting a radiation-responsive expression cassette composed of the promoter of early growth response-1 (Egr-1) and the pro-apoptotic protein TRAIL. We showed that the addition of adenovirus containing Egr-1/TRAIL significantly increased cell death and apoptosis caused by radiotherapy. In mice bearing xenograft tumors, intratumoral administration of the Egr-1/TRAIL adenovirus followed by radiation significantly reduced tumor growth and enhanced tumor survival. Our Egr-1/TRAIL adenoviral gene product may offer a novel “one-two punch” tumor therapy for cervical cancers not only by potentiating radiation treatment but also by preserving p53 defect-specific tumor killing of the oncolytic adenovirus.

A strong CTL response is dependent upon a high level of expression of specific class I major histocompatibility complex (MHC)/peptide complexes at the cell surface. An epitope-linked beta2-microglobulin (beta2m) molecule could provide a simple and more efficient means to enhance the formation of defined MHC/peptide complexes. However, the ability of an epitope-linked beta2m molecule to elicit primary CTL responses in vivo is still unknown. In this study, we modified the P1A tumor cell vaccine by addition of the tumor-associated epitope (TAE)-linked beta2m molecule and co-stimulatory molecule CD80 to improve the efficiency in the application of the vaccine. A eukaryotic co-expression vector consisting of the P1A35-43-linked beta2m molecule and the murine CD80 gene was constructed. P815 cell lines stably expressing P1A35-43-linked beta2m molecule and/or CD80 were established after transfection, by selection under G418. Administration of these inactivated tumor cell vaccines allowed the TAE-specific CD8+ T cell responses to be examined in vivo. Our results indicate that immunization with P815 cells expressing both the P1A35-43-linked beta2m molecule and the murine CD80 gene elicited a significantly stronger antitumor immune response than the single-modified tumor cell vaccines (expressing either P1A35-43-linked beta2m or CD80 alone). These findings support the feasibility and effectiveness of developing a dual-modified tumor cell vaccine consisting of the epitope-linked beta2m molecule and a co-stimulatory molecule.

Targeting tumor-related overexpression of anti-apoptotic Bcl2 protein by RNAi has been suggested as a potential treatment for cancer. However, the stability of RNAi and its delivery are still major obstacles to the clinical testing of Bcl2 RNAi. Here, we explore a novel strategy of expressing a synthetic Bcl2 microRNA (smRNA) in the 3' untranslated region (UTR) of the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), an apoptosis-inducing protein without apparent toxic effects in normal cells. TRAIL was specifically expressed from the human telomerase reverse transcriptase promoter (pTRT) that is active in many human tumors. Using this approach, we demonstrated that pTRT drove the tumor-specific expression of Bcl2 smRNA, which was processed by the host RNAi machinery and silenced endogenous Bcl2 expression in tumor cells. Bcl2 smRNA induced tumor cell apoptosis by activating caspase-3 and led to significant sensitization of tumor cells to TRAIL-induced apoptosis, while normal cells were spared. We also showed that the combined therapy of TRAIL-induced apoptosis and Bcl2 downregulation was superior to the mono-therapy of TRAIL or Bcl2 smRNA alone. This study proves a general paradigm for cancer therapy by using 3' UTR microRNA technology.

T cell-mediated cell immunity is the main anti-tumor immunity in which the effector T cells need specific antigen and costimulatory signals. One of the vaccines applied in tumor immunotherapy is the gene-modified tumor cell vaccine. One potential method to increase cell epitope density is to link the antigen with the major histcompatibility complex subunit beta2m. Our previous research indicated that the strategy of epitope fusion gene OVA-linker-beta2m can promote the formation of specific compounds on the tumor cell surface in vitro. In this study, we constructed two coexpression vectors pGL3-CD80-OVA-linker-beta2m and pGL3-IL21-OVA-linker-beta2m, in order to explore the cooperative action of CD80 or interleukin-21 (IL21) with the epitope fusion gene in anti-tumor immunity. Results showed that gene-modified B16 cells (B16/OVA, B16/CD80-OVA and B16/IL21-OVA) grew slower than B16 cells in vitro and in vivo, especially the B16/IL21-OVA subline, which illustrated that such gene modification decreased oncogenicity of malignant tumor cells. On the other hand, gene-modified tumor cell subline immunization can induce effective long-term anti-tumor immunity defending tumor cell attacks. IL21 played a more cooperative role with the OVA-linker-beta2m than CD80 in this study. This strategy might lay foundations for the research of a new type of tumor vaccine.

Bik, a BH3-only protein, was identified to induce cells apoptosis. In this study, we reported that Bik exclusively localized to endoplasmic reticulum rather than mitochondria. The apoptosis induced by Bik was inhibited in Hep3B cells, when TM domain of Bik was truncated. The ectopic overexpression of Bik protein caused the rapid and sustained elevation of the intracellular cytosolic Ca2+, which originated from the ER Ca2+ stores releasing. The Hep3B cells apoptosis induced by Bik was not prevented by establishing the clamped cytosolic Ca2+ condition, or by buffering of the extracellular Ca2+ with EGTA, suggesting that the depletion of ER Ca2+ stores rather than the elevation of cytosolic Ca2+ or the extracellular Ca2+ entry contributed to Bik-induced Hep3B cells apoptosis.

BIK, a member of the Bcl-2 family, has been reported to induce cell apoptosis but the underlying mechanisms are not well delineated. We used siRNA targeting caspase-12 to examine the effect of caspase-12 on apoptosis induced by BIK. In this study, we show that caspase-12 was activated by BIK. With caspase-12 knocked down, the apoptosis induced by BIK was decreased substantially. The activation of caspase-9 and depolarization of mitochondrial membrane potential were induced by BIK, which were decreased concomitant with caspase-12 silenced.

CD80, a costimulatory molecule, plays an important role in eliciting antitumor immunity. Without costimulation, recognition of antigens by T cells may not cause a response, even if tumor cells express MHC class I molecules and specific antigens. On the basis of the recombinant GFP-tagged Kb molecule, we constructed a co-expression vector of CD80 and GFP-tagged Kb molecules. The recombinant fusion was transfected into mouse melanoma B16 cells by electroporation; positive cells were obtained by G418 screening. Highly expressing monoclonal cells, irradiated by 137Cs, were used to immunize mice to obtain specific T cells, which were then cultivated with tumor cells in vitro and examined with a laser confocal microscope. The evident and intense uptake of the antigen peptide-MHC class I-GFP complex by specific T cells was visualized from the culture of B16/CD80-Kb-GFP and T cells. However, little uptake was observed from the culture of B16/Kb-GFP and T cells. These results show that co-expression of CD80 molecules with Kb, an MHC class I molecule, on the surface of B16 tumor cells can enhance the response of specific T cells and thus increase the uptake of the antigen peptide-MHC class I-GFP complex. The absorbed green fluorescence was concentrated mainly on the T cell surface, and this result might pave the way to eluting specific antigen peptides directly from T cells to find and isolate novel tumor-specific antigen peptides.

To specifically deliver the therapeutic gene to cancer cells and construct target retroviral vectors by inserting the single-chain variable antibody fragment into the retroviral envelope.Single-chain antibody expression vector pET -20bScfv was constructed. Binding activity of the genetically engineered single-chain variable antibody fragment was verified by enzyme-linked immunosorbent assay (ELISA) and Western blot. At the same time, by means of polymerase chain reaction (PCR)-directed mutagenesis, the appropriate cloning site EcoT22/Sal was generated at the N-terminus of receptor-binding SU domain in the MoMLV env polypeptide. Then the single- chain antibody gene, encoding a functional antibody, was inserted into the cloning site. The Scfv-env fusion gene fragment was subcloned into CMV expression vector. The Lac-Z retrovirus that co-displayed the Scfv-env chimeric protein and wild-type env protein was produced by transfection of Psi 2 cells with retroviral plasmid and the fusion gene expressing plasmid. To confirm the specificity of the recombinant retrovirus, infection assays and competitive inhibition assays were performed.The results of ELISA and Western blot showed that the genetically engineered single-chain variable antibody fragment could bind to the SHG44 cells surface membrane antigen. Virus-binding assay, viral infection and competitive inhibition assays confirmed that the harvested virus efficiently bound to and infected SHG44 cancer cells expressing the relative membrane antigen specially via the recognition of the target antigen.These results imply that insertion of Scfv into the retroviral envelope can specifically deliver the interested gene into specific antigen-producing cancer cells.

Background: The Toll-like receptor 2 (TLR2)-driven tissue response may promote neoangiogenesis and tumour growth by mechanisms that are poorly understood. Methods: We investigated the expression levels of TLR2 and associated-miRNAs in colorectal carcinoma (CRC) tissues and cell lines using real-time PCR, northern blotting and western blotting. Survival curver was generated by Log-Rank test and the role of TLR2 signalling in tumour invasion and migration was determined by transwell analysis kits. Results: We observed that the tissues from CRC patients express relatively high levels of TLR2. Targeting TLR2 markedly reduces the invasion and migration of CRC cells. We also found that miR-143, a putative tumour suppressor that is down-regulated in CRC tissues, reduces the invasion and migration of CRC cells primarily via TLR2. Utilising a xenograft mouse model, we demonstrated that re-expression of miR-143 inhibits CRC cell colonisation in vivo. Conclusion: miR-143 blocks the TLR2 signalling pathway in human CRC cells. This knowledge may pave the way for new clinical applications utilising miR-143 mimics in the treatment of patients with CRC.

Notch signaling pathway plays an important role in tumorigenesis by maintaining the activity of self-renewal of cancer stem cells, and therefore, it is hypothesized that interference of Notch signaling may inhibit tumor formation and progression. H101 is a recombinant oncolytic adenovirus that is cytolytic in cells lacking intact p53, but it is unable to eradicate caner stem cells. In this study, we tested a new strategy of tumor gene therapy by combining a Notch1-siRNA with H101 oncolytic adenovirus. In HeLa-S3 tumor cells, the combined therapy blocked the Notch pathway and induced apoptosis in tumors that are p53-inactive. In nude mice bearing xenograft tumors derived from HeLa-S3 cells, the combination of H101/Notch1-siRNA therapies inhibited tumor growth. Moreover, Notch1-siRNA increased Hexon gene expression at both the transcriptional and the translational levels, and promoted H101 replication in tumors, thereby enhancing the oncolytic activity of H101. These data demonstrate the feasibility to combine H101 p53-targted oncolysis and anti-Notch siRNA activities as a novel anti-cancer therapy.