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ARL-17477 is a selective neuronal nitric oxide synthase (NOS1) inhibitor that has been used in many preclinical studies since its initial discovery in the 1990s. In the present study, we demonstrate that ARL-17477 exhibits a NOS1-independent pharmacological activity that involves inhibition of the autophagy-lysosomal system and prevents cancer growth in vitro and in vivo. Initially, we screened a chemical compound library for potential anticancer agents, and identified ARL-17477 with micromolar anticancer activity against a wide spectrum of cancers, preferentially affecting cancer stem-like cells and KRAS-mutant cancer cells. Interestingly, ARL-17477 also affected NOS1-knockout cells, suggesting the existence of a NOS1-independent anticancer mechanism. Analysis of cell signals and death markers revealed that LC3B-II, p62, and GABARAP-II protein levels were significantly increased by ARL-17477. Furthermore, ARL-17477 had a chemical structure similar to that of chloroquine, suggesting the inhibition of autophagic flux at the level of lysosomal fusion as an underlying anticancer mechanism. Consistently, ARL-17477 induced lysosomal membrane permeabilization, impaired protein aggregate clearance, and activated transcription factor EB and lysosomal biogenesis. Furthermore, in vivo ARL-17477 inhibited the tumor growth of KRAS-mutant cancer. Thus, ARL-17477 is a dual inhibitor of NOS1 and the autophagy-lysosomal system that could potentially be used as a cancer therapeutic. [ABSTRACT FROM AUTHOR]

Purpose: Exosomes and their cargo microRNAs play a significant role in various biological processes in cancer. We hypothesized that microRNAs in exosomes secreted by gefitinib-resistant lung cancer cells might induce resistant phenotypes in otherwise gefitinib-sensitive lung cancer cells. Methods: We isolated exosomes generated by the gefitinib-resistant human lung adenocarcinoma cell line PS-9/ZD. PC-9, which is a gefitinib-sensitive cell line, was treated with the PC-9/ZD exosomes, and these PC-9 cells were analyzed for cell proliferation after treatment with gefitinib. miRNA arrays were analyzed in PC-9 and PC-9/ZD cells, and we isolated microRNAs that were expressed at elevated levels in PC-9/ZD cells. Furthermore, we transfected these microRNAs into PC-9 cells and analyzed the effects on the cells' sensitivity to gefitinib. Results: Exosomes isolated from PC-9/ZD cells significantly increased the proliferation of PC-9 cells during gefitinib treatment. A microRNA array analysis showed that miR-564, miR-658, miR-3652, miR-3126-5p, miR-3682-3p and miR-6810-5p were significantly upregulated in PC-9/ZD cells. PC-9 cells transfected with miR-564 or miR-658 showed chemo-resistant phenotypes. Conclusion: Exosomal miR-564 and miR-658 derived from gefitinib-resistant lung cancer cells induce drug resistance in sensitive cells. Cell-to-cell interaction via exosomal microRNAs may be a novel mechanism and therapeutic target of resistance against gefitinib. [ABSTRACT FROM AUTHOR]

During a previous study that aimed to identify anticancer agents within primate-consumed plants, the present group identified that Eugenia aquea (E. aquea) possessed potential as a source of anticancer agents. The ethanol extract of E. aquea leaves exhibited strong inhibitory activity against the proliferation of the human breast adenocarcinoma MCF-7 cell line. The inhibition of proliferation was determined using an MTT assay. The present study was performed to isolate the active compound within the E. aquea leaves that generated the aforementioned activity, and resulted in the isolation of 2',4'-dihydroxy-6-methoxy-3,5-dimethylchalcone, which was identified through the analysis of spectroscopic data. This compound was examined for its inhibitory activity against the MCF-7 cell line using a MTT assay, and the ability of 2',4'-dihydroxy-6-methoxy-3,5-dimethylchalcone to induce apoptosis through the activation of the poly(adenosine diphosphate-ribose) polymerase (PARP) protein was also investigated. The results of the present study revealed that the isolated compound inhibited cell proliferation in a dose-dependent manner, possessed an IC50 of 74.5 μg/ml (250 μM) and promoted apoptosis via the activation of PARP. It was concluded that these results indicated a requirement for additional investigations into 2',4'-dihydroxy-6-methoxy-3,5-dimethylchalcone in order to provide a basis for the use of this compound in the management of cancer. [ABSTRACT FROM AUTHOR]

Hu antigen R (HuR) regulates stress responses through stabilizing and/or facilitating the translation of target mRNAs. The human TRA2β gene encodes splicing factor transformer 2β (Tra2β) and generates 5 mRNA isoforms (TRA2β1 to -5) through alternative splicing. Exposure of HCT116 colon cancer cells to sodium arsenite stimulated checkpoint kinase 2 (Chk2)- and mitogen-activated protein kinase p38 (p38MAPK)-mediated phosphorylation of HuR at positions S88 and T118. This induced an association between HuR and the 39-nucleotide (nt) proximal region of TRA2β exon 2, generating a TRA2β4 mRNA that includes exon 2, which has multiple premature stop codons. HuR knockdown or Chk2/p38MAPK double knockdown inhibited the arsenite-stimulated production of TRA2β4 and increased Tra2β protein, facilitating Tra2β-dependent inclusion of exons in target pre-mRNAs. The effects of HuR knockdown or Chk2/p38MAPK double knockdown were also confirmed using a TRA2β minigene spanning exons 1 to 4, and the effects disappeared when the 39-nt region was deleted from the minigene. In endogenous HuR knockdown cells, the overexpression of a HuR mutant that could not be phosphorylated (with changes of serine to alanine at position 88 [S88A], S100A, and T118A) blocked the associated TRA2β4 interaction and TRA2β4 generation, while the overexpression of a phosphomimetic HuR (with mutations S88D, S100D, and T118D) restored the TRA2β4-related activities. Our findings revealed the potential role of nuclear HuR in the regulation of alternative splicing programs under oxidative stress. [ABSTRACT FROM AUTHOR]

Purpose:18F-FAMT as an amino-acid tracer for positron emission tomography (PET) is useful for detecting human neoplasms. 18F-FAMT is accumulated in tumour cells solely via L-type amino-acid transporter 1 (LAT1). This study was conducted to investigate the biological significance of 18F-FAMT uptake in patients with oesophageal cancer.Methods:From April 2008 to December 2011, 42 patients with oesophageal cancer underwent both 18F-FAMT PET/CT and 18F-FDG PET/CT before surgical treatment. The immunohistochemical analysis of LAT1, CD98, Ki-67, CD34, p53, p-Akt and p-mTOR was performed on the primary lesions. In vitro experiments were performed to examine the mechanism of 18F-FAMT uptake.Results:High uptake of 18F-FAMT was significantly associated with advanced stage, lymph node metastasis and the expression of LAT1, CD98, Ki-67 and CD34. LAT1 expression yielded a statistically significant correlation with CD98 expression, cell proliferation, angiogenesis and glucose metabolism. In vitro experiments revealed that 18F-FAMT was specifically transported by LAT1.Conclusions:The uptake of 18F-FAMT within tumour cells is determined by the LAT1 expression and correlated with cell proliferation and angiogenesis in oesophageal cancer. The present experiments also confirmed the presence of LAT1 as an underlying mechanism of 18F-FAMT accumulation. [ABSTRACT FROM AUTHOR]

Serine/arginine-rich splicing factor 3 (SRSF3) is a member of the SR protein family and plays wide-ranging roles in gene expression. The human SRSF3 gene generates two alternative splice transcripts, a major mRNA isoform (SRSF3-FL) encoding functional full-length protein and a premature termination codon (PTC)-containing isoform (SRSF3-PTC). The latter is degraded through nonsense-mediated mRNA decay (NMD). Treatment of a human colon cancer cell line (HCT116) with 100 µM sodium arsenite increased SRSF3-PTC mRNA levels without changing SRSF3-FL mRNA levels. A chemiluminescence- based NMD reporter assay system demonstrated that arsenite treatment inhibited NMD activity and increased SRSF3-PTC mRNA levels in the cytoplasm, facilitating translation of a truncated SRSF3 protein (SRSF3-TR) from SRSF3-PTC mRNA. SRSF3-TR lacked two-thirds of the Arg/Ser-rich (RS) domain whose phosphorylation state is known to be crucial for subcellular distribution. SRSF3-FL was localized in the nucleus, while overexpressed SRSF3-TR was diffusely distributed in the cytoplasm and the nucleus. A part of SRSF3-TR was also associated with stress granules in the cytoplasm. Interestingly, treatment of HCT116 cells with a small interference RNA specifically targeting SRSF3-PTC mRNA significantly attenuated arsenite-stimulated induction of c-JUN protein, its binding activity to the AP-1 binding site (-126 to 120 bp) in the interleukin (IL)-8 gene promoter, and AP-1 promoter activity, resulting in significant reduction of arsenite-stimulated IL-8 production. Our results suggest that SRSF3-TR may function as a positive regulator of oxidative stress-initiated inflammatory responses in colon cancer cells. [ABSTRACT FROM AUTHOR]

Background: Transformer (Tra) 2β is a member of the serine/arginine-rich (SR)-like protein family that regulates alternative splicing of numerous genes in a concentration-dependent manner. Several types of cancer cells up-regulate Tra2β expression, while the regulatory mechanism of Tra2β expression remains to be elucidated. In this study, we examined the transcriptional regulation and possible functions of Tra2β in human colon cancer cells. Methods: We cloned 959 bp-upstream of the human TRA2β 5′-flank into luciferase constructs. Chromatin immunoprecipitation (ChIP) was employed to identify crucial cis element(s) and trans activator(s) of the TRA2β promoter. Tra2β expression in the human colon and colon cancer tissues was examined by immunohistochemistry. Results: In response to sodium arsenite, colon cancer cells (HCT116) increased levels of TRA2β1 mRNA encoding a functional, full-length Tra2β with a peak around 6 h without changing its mRNA stability. Transient expression assays using a reporter gene driven by serially truncated TRA2β promoters and Chip assay demonstrated that an Ets1-binding site present at −64 to −55 bp was crucial for basal transcription, while three heat shock elements (HSEs) located at −145 to −99 bp mediated the oxidant-induced transactivation of TRA2β. Tra2β knockdown caused apoptosis of HCT116 cells. Tra2β were preferentially expressed in proliferative compartment of normal human colonic glands and adenocarcinomas, where Ets1 and heat shock factor 1 were also highly expressed. Conclusions: Our results suggest that oxidative stress-responsive Tra2β may play an important role in colon cancer growth. [ABSTRACT FROM AUTHOR]

Background: Vascular endothelial growth factor-a (VEGF)-targeted therapies have become an important treatment for a number of human malignancies. The VEGF inhibitors are actually effective in several types of cancers, however, the benefits are transiently, and the vast majority of patients who initially respond to the therapies will develop resistance. One of possible mechanisms for the acquired resistance may be the direct effect(s) of VEGF inhibitors on tumor cells expressing VEGF receptors (VEGFR). Thus, we investigated here the direct effect of chronic VEGF inhibition on phenotype changes in human colorectal cancer (CRC) cells. Methods: To chronically inhibit cancer cell-derived VEGF, human CRC cell lines (HCT116 and RKO) were chronically exposed (2 months) to an anti-VEGF monoclonal antibody (mAb) or were disrupted the Vegf gene (VEGF-KO). Effects of VEGF family members were blocked by treatment with a VEGF receptor tyrosine kinase inhibitor (VEGFRTKI). Hypoxia-induced apoptosis under VEGF inhibited conditions was measured by TUNEL assay. Spheroid formation ability was assessed using a 3-D spheroid cell culture system. Results: Chronic inhibition of secreted/extracellular VEGF by an anti-VEGF mAb redundantly increased VEGF family member (PlGF, VEGFR1 and VEGFR2), induced a resistance to hypoxia-induced apoptosis, and increased spheroid formation ability. This apoptotic resistance was partially abrogated by a VEGFR-TKI, which blocked the compensate pathway consisted of VEGF family members, or by knockdown of Vegf mRNA, which inhibited intracellular function (s) of all Vegf gene products. Interestingly, chronic and complete depletion of all Vegf gene products by Vegf gene knockout further augmented these phenotypes in the compensate pathway-independent manner. These accelerated phenotypes were significantly suppressed by knockdown of hypoxia-inducible factor-1α that was upregulated in the VEGF-KO cell lines. Conclusions: Our findings suggest that chronic inhibition of tumor cell-derived VEGF accelerates tumor cell malignant phenotypes [ABSTRACT FROM AUTHOR]

Background: Drug resistance in colorectal cancers is assumed to be mediated by changes in the expression of microRNAs, but the specific identities and roles of microRNAs are largely unclear. We examined the effect of 5-fluorouracil (5-FU) resistance on microRNA expression. Methods: Two types of 5-FU-resistant colon cancer cells were derived from the DLD-1 and KM12C cell lines. The expressions of microRNAs were profiled with a microarray containing 723 microRNAs and validated by quantitative real-time polymerase chain reaction (qRT-PCR). To survey the downstream mediators of microRNA, we used a microRNA:mRNA immunoprecipitation (RIP)-Chip and pathway analysis tool to identify potential direct targets of microRNA. Results: In response to 5-FU, miR-19b and miR-21 were over-expressed in 5-FU-resistant cells. Of note, miR-19b was up-regulated 3.47-fold in the DLD-1 resistant cells, which exhibited no alteration in cell cycle profiles despite exposure to 5-FU. After transfection of miR-19b, specific mRNAs were recruited to microRNA:mRNA complexes isolated with Ago2 antibody and subjected to whole-genome transcriptional analysis. In this analysis, 66 target mRNAs were enriched by at least 5.0-fold in the microRNA:mRNA complexes from DLD-1 resistant cells. Ingenuity pathway analysis of mRNA targets significantly ( P < 0.05) indicated the category 'Cell Cycle' as a probable area of the molecular and cellular function related with 5-FU resistance. Among candidate mRNA targets, SFPQ and MYBL2 have been linked to cell cycle functions. Conclusions: We revealed up-regulation of miR-19b in response to 5-FU and potential targets of miR-19b mediating the cell cycle under treatment with 5-FU. Our study provides an important insight into the mechanism of 5-FU resistance in colorectal cancers. [ABSTRACT FROM AUTHOR]

Our recent study demonstrated that higher expression of N-myc downregulated gene 1 (NDRG1) is closely correlated with poor prognosis in gastric cancer patients. In this study, we asked whether NDRG1 has pivotal roles in malignant progression including metastasis of gastric cancer cells. By gene expression microarray analysis expression of NDRG1 showed the higher increase among a total of 3691 up-regulated genes in a highly metastatic gastric cancer cell line (58As1) than their parental low metastatic counterpart (HSC-58). The highly metastatic cell lines showed decreased expression of E-cadherin, together with enhanced expression of vimentin and Snail. This decreased expression of E-cadherin was restored by Snail knockdown in highly metastatic cell lines. We next established stable NDRG1 knockdown cell lines (As1/Sic50 and As1/Sic54) from the highly metastatic cell line, and both of these cell lines showed enhanced expression of E-cadherin and decreased expression of vimentin and Snail. And also, E-cadherin promoter-driven luciferase activity was found to be increased by NDRG1 knockdown in the highly metastatic cell line. NDRG1 knockdown in gastric cancer cell showed suppressed invasion of cancer cells into surround tissues, suppressed metastasis to the peritoneum and decreased ascites accumulation in mice with significantly improved survival rates. This is the first study to demonstrate that NDRG1 plays its pivotal role in the malignant progression of gastric cancer through epithelial mesenchymal transition. [ABSTRACT FROM AUTHOR]

Background: We previously demonstrated that extracapsular invasion (ECI) at a metastatic sentinel node was significantly associated with the presence of positive non-sentinel nodes in patients with breast cancer. However, the mechanism of metastatic spreading of tumor cells to distant lymph nodes in patients with colorectal carcinoma is not fully understood. In this study, we investigated the factors that may determine the likelihood of additional regional lymph node metastasis when metastasis is found in nodes at the N1 site in colorectal cancer, especially focusing on the presence of ECI. Methods: Two hundred and twenty-eight consecutive patients who underwent colorectal resection were identified for inclusion in this study, of which 37 (16.2%) had positive lymph nodes at the N1 site. Six of these 37 cases had additional metastasis in N2 site lymph nodes. We reviewed the clinicopathological features of these cases and performed statistical analysis of the data. Results: In the univariate analysis ECI at the N1 site was the only factor significantly associated with the presence of cancer cells in the N2 site. Other factors, including number of positive lymph nodes, lymphovascular invasion of the primary tumor, tumor size and tumor depth of invasion, were not associated with metastatic involvement at the N2 site. Conclusions: Our results suggest that the presence of ECI at metastatic lymph nodes at the N1 site is correlated with further metastasis at the N2 site. These findings imply the possibility that ECI might indicate the ability of colorectal tumor cells to disseminate to distant lymph nodes. [ABSTRACT FROM AUTHOR]

Abstract  We have reported that the levels of the soluble molecule of the human leukocyte antigen class I (sHLA-I) in patients with advanced gastric cancer were significantly lower than those in patients with cancer in the early stages. However, the effect of sHLA-I on gastric cancer cells has not been elucidated. Using human gastric cancer cell lines, MKN28, MKN45, and MKN74, we evaluated the effects of sHLA-I on cell growth, DNA synthesis, and apoptosis induction. Three types of synthesized peptides derived from HLA-I were also examined for their capacity to induce apoptosis. sHLA-I and a synthesized peptide, nos. 220–232 of the α3 domain of HLA-B7, caused cell growth inhibition by inducing apoptosis in human gastric cancer cells. This peptide also inhibited the in vivo growth of cancer dissemination caused by an intraperitoneal injection of MKN45 into severe combined immunodeficient mice. In conclusion, sHLA-I and the peptides derived from HLA-I cause apoptosis in human gastric cancer cell lines. [ABSTRACT FROM AUTHOR]

Background: Expression of the tumor suppressor p16INK4a increases during aging and replicative senescence. Methodology/Principal Findings: Here, we report that the microRNA miR-24 suppresses p16 expression in human diploid fibroblasts and cervical carcinoma cells. Increased p16 expression with replicative senescence was associated with decreased levels of miR-24, a microRNA that was predicted to associate with the p16 mRNA coding and 3'-untranslated regions. Ectopic miR-24 overexpression reduced p16 protein but not p16 mRNA levels. Conversely, introduction of antisense (AS)-miR-24 blocked miR-24 expression and markedly enhanced p16 protein levels, p16 translation, and the production of EGFP-p16 reporter bearing the miR-24 target recognition sites. Conclusions/Significance: Together, our results suggest that miR-24 represses the initiation and elongation phases of p16 translation. [ABSTRACT FROM AUTHOR]

Y-box-binding protein 1 (YB-1), which is a member of the DNA-binding protein family containing a cold-shock domain, has pleiotropic functions in response to various environmental stimuli. As we previously showed that YB-1 is a global marker of multidrug resistance in ovarian cancer and other tumor types. To identify YB-1-regulated genes in ovarian cancers, we investigated the expression profile of YB-1 small-interfering RNA (siRNA)-transfected ovarian cancer cells using a high-density oligonucleotide array. YB-1 knockdown by siRNA upregulated 344 genes, including MDR1, thymidylate synthetase, S100 calcium binding protein and cyclin B, and downregulated 534 genes, including CXCR4, N-myc downstream regulated gene 1, E-cadherin and phospholipase C. Exogenous serum addition stimulated YB-1 translocation from the cytoplasm to the nucleus, and treatment with Akt inhibitors as well as Akt siRNA and integrin-linked kinase (ILK) siRNA specifically blocked YB-1 nuclear localization. Inhibition of Akt activation downregulated CXCR4 and upregulated MDR1 (ABCB1) gene expression. Administration of Akt inhibitor resulted in decrease in nuclear YB-1-positive cancer cells in a xenograft animal model. Akt activation thus regulates the nuclear translocation of YB-1, affecting the expression of drug-resistance genes and other genes associated with the malignant characteristics in ovarian cancer cells. Therefore, the Akt pathway could be a novel target of disrupting the nuclear translocation of YB-1 that has important implications for further development of therapeutic strategy against ovarian cancers.Oncogene (2007) 26, 2736–2746. doi:10.1038/sj.onc.1210084; published online 30 October 2006 [ABSTRACT FROM AUTHOR]

We investigated the effects of pegylated IFN-α2b (PEG-IFN-α2b) on the growth of human liver cancer cells. Methods: The effect of PEG-IFN-α2b on the proliferation of 13 liver cancer cell lines was investigated in vitro. Chronological changes in growth and IFN-α receptor-2 (IFNAR-2) expression were monitored in hepatocellular carcinoma (HCC) cells (HAK-1B) cultured with PEG-IFN-α2b. After HAK-1B cells were transplanted into nude mice, various doses of PEG-IFN-α2b or IFN-α2b were administered, and tumor volume, weight, histology, and IFNAR-2 expression were examined. Results: PEG-IFN-α2b inhibited the growth of nine cell lines with apoptosis in a dose- and time-dependent manner. Continuous contact with PEG-IFN-α2b induced time-dependent growth inhibition and down-regulation of IFNAR-2 expression. PEG-IFN-α2b induced a dose-dependent decrease in tumor volume and weight, a significant increase of apoptotic cells, and a decrease in IFNAR-2 expression in the tumor. The clinical dose for chronic hepatitis C was also effective. The antitumor effect of PEG-IFN-α2b was significantly stronger than that of non-PEG-IFN-α2b in vivo. Conclusions: Continuous contact with PEG-IFN-α2b induces strong antitumor effects and the down-regulation of IFNAR-2 in HCC cells. The data suggest potential clinical application of PEG-IFN-α2b for the prevention and treatment of HCC. [ABSTRACT FROM AUTHOR]

Objective: Although cell density in cultured cells has demonstrated several alterations in the nature of cell kinetics, the changes in the metastatic aggressiveness of cancer lines under different cell densities have not yet been studied. Methods: In the current study, we investigated the influence of changing the cell density of cultured cancer cells (colon 26 and B16-F10) injected into the tail vein in BALB/c mice on the metastatic activity by evaluating the number of lung metastases, and the possible mechanisms of this phenomenon were discussed based on the basis of the results of an invasion assay and a cell adhesion assay. Results: The number of metastatic nodules was significantly higher in the high-density group than in the low one in colon 26 (p < 0.005), however, this phenomenon was not seen in B16-F10. Next, we performed the same experiment by changing the environment to the opposite conditions for the cells in the low- and high-density groups, and the results showed the metastatic activities to be always higher in the high-density group. Moreover, although no difference was seen regarding the invasive activity between the high- and low-density groups, an adhesion assay showed the difference in the adhesion cell rate to be significantly higher in the high-density group especially in early period after coculture with human umbilical vein endothelial cells (HUVEC) (p < 0.05). Conclusion: In some cell types, the metastatic activity could be altered and reversed by changing the environment, such as the cell density, during a relatively short period. As a result, the epigenetic changes of cancer cells are thus suggested to play a certain role in the malignant potentiality. Copyright © 2004 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Four kinds of human cancer cell lines and one mouse caner cell line were inoculated into the subepithelial area of the anorectum of female nude mice. Among the cell lines, two cell lines (KATO III and Lu 135) showed the potential enforcement of atypical changes in the adjacent mouse anorectal epithelium. Moreover, the submucosal invasion of the malignant transformed cells of the mouse epithelium was demonstrated in specimens obtained from three KATO III-inoculated mice. This exciting and novel phenomenon clearly demonstrates the need to change the present general concept of a single-cell origin of cancer tissue. This valuable and novel discovery may change the basis of oncology research while also providing new ideas for projects to investigate the mechanisms of carcinogenesis from several aspects such as molecular biology, cell biology, and pathology. Moreover, the novel experimental design itself is also extremely useful as a simple model for investigating the mechanisms of oncogenesis. [ABSTRACT FROM AUTHOR]

Methylation-associated inactivation of RASSF1, a putative tumor suppressor identified at 3p21.3, is reported in several cancers. We examined RASSF1 in non-small lung cancer (NSCLC) to search for clinical implications. RT-PCR analysis showed no expression of RASSF1A in 12 of 20 lung cancer cell lines. Loss of expression correlated well with promoter methylation status of these lines. Sequence analysis revealed 2 polymorphisms (codons 21 and 133) in RASSF1A transcripts, but not in RASSF1C transcripts. No somatic mutations were found. Of 7 cell lines with K-ras mutations at codon 12 or 61, 2 lost expression of RASSF1A, whereas in 13 cell lines with wild-type K-ras gene, 10 lost RASSF1A gene expression (p = 0.0521). We investigated methylation status of this putative tumor suppressor gene in 100 primary NSCLCs to determine whether there is a clinical significance. Forty-two of primary NSCLCs demonstrated methylated allele. There is no correlation between promoter methylation of RASSF1A and clinicopathological findings, including histological type or grade, tumor staging, p53 and K-ras mutational status, or patients' survival. In the cases of Stage I and II disease, however, RASSF1A methylation was associated with earlier recurrence (p = 0.0247). Epigenetic silencing of RASSF1A is a frequent event in non-small lung cancer and will provide novel opportunities to develop diagnosis and therapy of NSCLC. © 2003 Wiley-Liss, Inc. [ABSTRACT FROM AUTHOR]

We previously identified 9 genes (i.e., thymosin β4, secreted protein acidic and rich in cysteine, Cap43, ceruloplasmin, serum amyloid A, heat shock protein 90, LOT1, osteopontin and casein kinase Iγ) that are more highly expressed in cancerous regions than in noncancerous regions in human renal cancers. In our study, we considered the possibility that the von Hippel-Lindau (VHL) tumor suppressor gene might be able to affect the expression of these 9 genes in renal cancer cells. We first established 2 VHL-positive cell lines, 786/VHL-1 and 786/VHL-2, after the introduction of wild-type VHL into VHL-negative renal cancer 786-O cells. Of these 9 genes, expression of the Cap43 gene was specifically downregulated by VHL. Expression of Cap43 was also much lower in 4 other VHL-positive renal cancer cell lines than in VHL-negative 786-O cells. Cap43 promoter assays with several deletion or mutation constructs demonstrated that the Sp1 site in the element from –286 base pairs (bp) to –62 bp was partly responsible for VHL-induced suppression of the Cap43 gene. Immunostaining analysis with human specimens of renal cancers demonstrated that the Cap43 protein was expressed in most cancer cells and macrophages. We also observed a marked and specific increase of Cap43 mRNA levels in response to hypoxia or nickel in all VHL-positive cell lines. Cellular expression of Cap43 mRNA in response to hypoxia or nickel thus is closely associated with VHL gene expression in renal cancer cells. Although the function of the Cap43 protein remains unclear, the expression of Cap43 protein could be a molecular marker closely associated with VHL in renal cancer. © 2003 Wiley-Liss, Inc. [ABSTRACT FROM AUTHOR]

PURPOSE: Recent advances have made possible the treatment of small invasive colorectal cancer by means of polypectomy or endoscopic mucosal resection. CD44 expression in cancer cells was identified as an indicator of lymph-node metastasis, which could be evaluated in specimens removed by colonoscopy. METHODS: The correlation between lymph-node metastasis and the expression of standard-type CD44 in cancer cells was examined immunohistologically using the invaded cancer cells of 61 tissue samples of superficially invasive colorectal cancer. We defined the above as invasive cancer restricted within the colorectal wall. Of the 61 samples, 31 had submucosal invasion and 30 had muscular invasion. RESULTS: Standard-type CD44 expression in the area of invasion in cases with lymph-node metastasis was remarkably down-regulated. In 43 cases with no lymph-node metastasis, 36 (83.7 percent) of patients had CD44 expression in invaded cells, whereas only two of 18 cases (11.1 percent) with lymph-node metastasis had expression of standard-type CD44 in the same area (P < 0.0001). A total of 69 6 percent (16/23) of patients with loss of standard-type CD44 expression in invaded sites were found to have positive metastasis in the lymph nodes. These results suggest that standard-type CD44 in invasive colon cancer cells could suppress metastasis to the regional lymph nodes. CONCLUSION; In cases of invasive colorectal cancer, the loss of standard-type CD44 expression in the invaded area is a sensitive marker for metastasis to the lymph nodes. Further investigation with larger patient groups is required to clarify the reliability of loss of standard-type CD44 expression as an indicator for additional surgery after endoscopic resection of submucosal invasive colorectal carcinoma. [ABSTRACT FROM AUTHOR]

The levels of hypoxia-inducible factor 1α (HIF-1α) are tightly controlled. Here, we investigated the posttranscriptional regulation of HIF-1α expression in human cervical carcinoma HeLa cells responding to the hypoxia mimetic CoCl2. Undetectable in untreated cells, HIF-1α levels increased dramatically in CoCl2-treated cells, while HIF-1α mRNA levels were unchanged. HIF-1α translation was potently elevated by CoCl2 treatment, as determined by de novo translation analysis and by monitoring the polysomal association of HIF-1α mRNA. An internal ribosome entry site in the HIF-1α 5' untranslated region (UTR) was found to enhance translation constitutively, but it did not further induce translation in response to CoCl2 treatment. Instead, we postulated that RNA-binding proteins HuR and PTB, previously shown to bind HIF-1α mRNA, participated in its translational upregulation after CoCl2 treatment. Indeed, both RNA-binding proteins were found to bind HIF-1α mRNA in a CoCl2-inducible manner as assessed by immunoprecipitation of endogenous ribonucleoprotein complexes. Using a chimeric reporter, polypyrimidine tract-binding protein (PTB) was found to bind the HIF-1α 3'UTR, while HuR associated principally with the 5'UTR. Lowering PTB expression or HuR expression using RNA interference reduced HIF-1α translation and expression levels but not HIF-1α mRNA abundance. Conversely, HIF-1α expression and translation in response to CoCl2 were markedly elevated after HuR overexpression. We propose that HuR and PTB jointly upregulate HIF-1α translation in response to CoCl2. [ABSTRACT FROM AUTHOR]

The expression of N-myc downstream-regulated gene 1 (NDRG1) was significantly correlated with tumor angiogenesis and malignant progression together with poor prognosis in gastric cancer. However, the underlying mechanism for the role of NDRG1 in the malignant progression of gastric cancer remains unknown. Here we examined whether and how NDRG1 could modulate tumor angiogenesis by human gastric cancer cells. We established NU/Cap12 and NU/Cap32 cells overexpressing NDRG1 inNUGC-3 cells, which show lower tumor angiogenesis in vivo. Compared with parental NU/Mock3, NU/Cap12, and NU/Cap32 cells: 1) induced higher tumor angiogenesis than NU/Mock3 cells accompanied by infiltration of tumor-associated macrophages in mouse dorsal air sac assay and Matrigel plug assay; 2) showed much higher expression of CXC chemokines, MMP-1, and the potent angiogenic factor VEGF-A; 3) increased the expression of the representative inflammatory cytokine, IL-1α 4) augmented JNK phosphorylation and nuclear expression of activator protein 1 (AP-1). Further analysis demonstrated that knockdown of AP-1 (Jun and/or Fos) resulted in down-regulation of the expression of VEGF-A, CXC chemokines, and MMP-1, and also suppressed expression of IL-1α in NDRG1-overexpressing cell lines. Treatment with IL-1 receptor antagonist (IL-1ra) resulted in down-regulation of JNK and c-Jun phosphorylation, and the expression of VEGF-A, CXC chemokines, and MMP-1 in NU/Cap12 and NU/Cap32 cells. Finally, administration of IL-1ra suppressed both tumor angiogenesis and infiltration of macrophages by NU/Cap12 in vivo. Together, activation of JNK/AP-1 thus seems to promote tumor angiogenesis in relationship to NDRG1-induced inflammatory stimuli by gastric cancer cells. [ABSTRACT FROM AUTHOR]

Abstract: N-myc downstream regulated gene-1 (NDRG1)/Cap43 regulates tumor growth and metastasis in various carcinomas. In this study we examined whether and how NDRG1/Cap43 modulates tumor growth by human hepatocellular carcinoma (HCC) cells. NDRG1/Cap43 cDNA was used to transfect HCC cell lines (KIM-1), and stable transfectants overexpressing NDRG1/Cap43 (KIM-1/Cap43) were obtained. Cell cycle analysis showed that KIM-1/Cap43 cells were arrested in the G0/G1 phase. Western blot analysis demonstrated an increase in p21 in KIM-1/Cap43 cells in culture under full confluency as compared with KIM-1/Mock. When KIM-1 cells, which are very low in NDRG1/Cap43 expression, were treated with mimosine, a G0/G1 cell cycle blocker, expression of NDRG1/Cap43 was induced in a dose dependent manner, together with p21 induction and CDK4 reduction. In vivo, KIM-1/Cap43 cells showed markedly decreased tumor growth rates compared with those of KIM-1/Mock. Immunohistochemical staining demonstrated markedly higher p21 labeling index in the KIM-1/Cap43 tumor than KIM-1/Mock tumor, and lower CDK4 and Ki-67 labeling index in the KIM-1/Cap43 than KIM-1/Mock. In order to confirm suppressive effects of NDRG1/Cap43, we further established a stable transfectant expressing NDRG1/Cap43 (HAK-1B/Cap43) using another HCC cell line, HAK-1B. Western blot analysis demonstrated an increase in p21 and a decrease in CDK4 in HAK-1B/Cap43 cells in culture under full confluency as compared with HAK-1B/Mock. HAK-1B/Cap43 also showed decreased tumor growth rates as compared with its control counterpart in vivo. NDRG1/Cap43 overexpression thus induced cell cycle arrest at the G0/G1 phase accompanied by increased p21 and decreased CDK4 expression in HCC cells. NDRG1/Cap43 might play a key role in the cell cycle control of G0/G1 in HCC cells. [Copyright &y& Elsevier]

The Y-box binding protein 1 (YB-1) is upregulated in many human malignancies including glioblastoma (GBM). It is also essential for normal brain development, suggesting that YB-1 is part of a neural stem cell (NSC) network. Here, we show that YB-1 was highly expressed in the subventricular zone (SVZ) of mouse fetal brain tissues but not in terminally differentiated primary astrocytes. Conversely, YB-1 knockout mice had reduced Sox-2, nestin, andmusashi-1 expression in the SVZ. Although primary murine neurospheres were rich in YB-1, its expression was lost during glial differentiation. Glial tumors often express NSC markers and tend to loose the cellular control that governs differentiation; therefore, we addressed whether YB-1 served a similar role in cancer cells. YB-1, Sox-2, musashi-1, Bmi-1, and nestin are coordinately expressed in SF188 cells and 9/9 GBM patient- derived primary brain tumor--initiating cells (BTIC). Silencing YB-1 with siRNA attenuated the expression of these NSC markers, reduced neurosphere growth, and triggered differentiation via coordinate loss of GSK3-b. Furthermore, differentiation of BTIC with 1% serum or bone morphogenetic protein-4 suppressed YB-1 protein expression. Likewise, YB-1 expression was lost during differentiation of normal human NSCs. Consistent with these observations, YB-1 expression increased with tumor grade (n=49 cases). YB-1 was also coexpressed with Bmi-1 (Spearmans 0.80, P > 0.001) and Sox-2 (Spearmans 0.66, P > 0.001) based on the analysis of 282 cases of high-grade gliomas. These proteins were highly expressed in 10/15 (67%) of GBM patients that subsequently relapsed. In conclusion, YB-1 correlatively expresses with NSC markers where it functions to promote cell growth and inhibit differentiation. Cancer Res; 71(16); 5569-78. ©2011 AACR. [ABSTRACT FROM AUTHOR]

Abstract: We have recently reported that N-myc downstream-regulated gene 1 (NDRG1)/Ca2+-associated protein with a molecular mass of 43kDa (Cap43) suppresses angiogenesis and tumor growth of pancreatic cancer through marked decreases in both the expression of CXC chemokines and phosphorylation of a NF-κB signaling molecule, inhibitor of κB kinase (IκBα). NDRG1/Cap43 is phosphorylated at serine/threonine sites in its C-terminal domain by serum- and glucocorticoid-regulated kinase 1 (SGK1). In this study, we attempted to clarify the domain or site of NDRG1/Cap43 responsible for its suppression of CXC chemokine expression in pancreatic cancer cells. Expression of the deletion constructs CapΔ2 [deletion of amino acids (AA) 130–142] and CapΔ4 [deletion of AA 180–294] as well as the wild-type full sequence of NDRG1/Cap43 (F-Cap), suppressed the production of CXC chemokines such as Groα/CXCL1 and ENA-78/CXCL5, whereas no or low suppression was observed in cell expressing the CapΔ5 mutant [deletion of AA 326–350] and CapΔ6 mutant [deletion of AA 326–394]. We further introduced mutations at the serine and threonine sites at 328 [T328A], 330 [S330A] and 346 [T346A], which are susceptible to phosphorylation by SGK1, and also constructed double mutants [T328A, S330A], [T328A, T346A] and [S330A, T346A]. Expression of all these mutants, with the exception of [S330A, T346A], suppressed the production of CXC chemokine to similar levels as their wild-type counterpart. IκBα was found to be specifically phosphorylated by this double mutant [S330A, T346A] and the CapΔ5 mutant at levels comparable to that induced in their wild-type counterpart. Phosphorylation of NDRG1/Cap43 at both serine330 and threonine346 is required for its suppressive action on the NF-κB signaling pathway and CXC chemokine expression in pancreatic cancer cells. [Copyright &y& Elsevier]

Y-box binding protein-1 (YB-1) plays pivotal roles in acquisition of global drug resistance and cell growth promotion through transcriptional activation of genes for both drug resistance and growth factor receptors. In this study, we investigated whether YB-1 is involved in regulation of the cell cycle and cell proliferation of human cancer cells. Treatment with YB-1 siRNA caused a marked suppression of cell proliferation and expression of a cell cycle related gene, CDC6 by cancer cells. Of cell cycle of cancer cells, S phase content was specifically reduced by knockdown of YB-1. The overexpression of CDC6 abrogated this inhibition of both cell proliferation and S phase entry. ChIP assay demonstrated that YB-1 binds to a Y-box located in the promoter region of the CDC6 gene. Expression of cyclin D1, CDK1 and CDK2 was also reduced with increased expression of p21Cip1 and p16INK4A when treated with YB-1 siRNA. Furthermore, the nuclear YB-1 expression was significantly associated with the level of CDC6 nuclear expression in patients with breast cancer. In conclusion, YB-1 plays an important role in cell cycle progression at G1/S of human cancer cells. YB-1 thus could be a potent biomarker for tumour growth and cell cycle in its close association with CDC6. [Copyright &y& Elsevier]

It is well known that phosphoglucose isomerase/autocrine motility factor (AMF) promotes cell migration in an autocrine manner in various tumor cells. However, it remains unclear whether certain cytokines modulate the effects of AMF on tumor cell migration. Because interleukin (IL)-8, a proinflammatory cytokine, is produced by melanoma cells and has been correlated with melanoma migration, the migratory ability of melanoma cells induced by AMF may also involve induction of IL-8 expression. In the present study, we assessed whether AMF promotes melanoma cell migration through autocrine production of IL-8. We found that AMF stimulation increased IL-8 production through up-regulation of IL-8 mRNA transcription, especially in biologically early stage melanoma cells. AMF-induced migration of these cells was inhibited by a specific neutralizing antibody against IL-8. The IL-8 production induced by AMF was mediated by the ERK1/2 pathways. These findings suggest that melanoma migration induced by AMF is mediated by autocrine production of IL-8 as a novel downstream modulator of the AMF signaling pathway. [ABSTRACT FROM AUTHOR]

Abstract: 5-FU is commonly used for treatment of various solid tumors including colon carcinoma. We have previously demonstrated that Egr-1 induced by 5-FU enhanced TSP-1 expression in human colon cancer KM12C cells. In this study, a Genechip analysis of KM12C cells treated with 5-FU revealed down-regulation of 924 genes and up-regulation of 460 genes. The decreased expression of c-Myc mRNA and phosphorylated c-Myc were detected and confirmed by RT-PCR and immunoblotting. Since 5-FU induced the expression of TSP-1, we examined the effect of c-Myc on the TSP-1 promoter. Deletion of the TSP-1 promoter region in which binding sites for c-Myc reside had no effect on the TSP-1 promoter activity induced by 5-FU. Meanwhile, 5-FU dose-dependently decreased the expression of miR-17-92 cluster. These findings suggest that 5-FU decreased the expression of c-Myc and consequently miR-17-92 cluster and increased the expression of TSP-1 mRNA. [Copyright &y& Elsevier]

Abstract: Objective. : Selective expression of beta-tubulin isotypes has been reported to be one of the important mechanisms of taxane resistance. The purpose of this study was to evaluate the immunohistochemical expression of beta-tubulin isotypes using clinical samples of ovarian carcinoma treated by taxanes and to examine whether the protein levels of each of the beta-tubulin isotypes were correlated with the clinical features. Experimental design. : We examined tumor samples taken from 77 ovarian carcinoma patients (54 patients treated with a taxane-based regimen and 23 treated with a taxane-free regimen), for the intrinsic protein level of beta-tubulin isotype (classes I, II, III and IV) expression using immunohistochemistry, and we evaluated the correlation of this protein level with the clinical features. The expression levels were scored by the proportion and intensity of the immunoreactive tumor cells. Results. : High protein levels of classes I and IV beta-tubulin, and very low protein levels of class II beta-tubulin, and intermediate protein levels of class III beta-tubulin expression were demonstrated in a total of 77 ovarian carcinomas. As for the samples taken from the 54 patients treated with the taxane-based regimen, 40 samples demonstrated undetectable levels of class II beta-tubulin protein. The class II beta-tubulin expression-absent group was significantly correlated with advanced stage (p =0.024) and with a short period of progression-free survival (log-rank test, p =0.022). Multivariate analyses demonstrated that the only significant independent prognostic indicator of a short period of progression-free survival was advanced stage, although a high expression of class III beta-tubulin was also prone to be associated with a short period of progression-free survival, but not significantly so (p =0.081). No such correlations or propensities were demonstrated in the 23 patients treated with the taxane-free regimen. Conclusions. : In cases of ovarian carcinoma treated by taxanes, high expression of class III beta-tubulin seems to be associated with earlier recurrence, which is believed likely to be resistant relapse. In addition, loss of class II beta-tubulin expression is correlated with advanced stage, which may represent aggressive tumor progression. [Copyright &y& Elsevier]

Abstract: There is growing evidence that Rho proteins are deregulated by overexpression in tumours; and according to some reports, this correlates with disease progression. Our previous clinical study had demonstrated a correlation between RhoA expression and tumour progression in oesophageal squamous cell carcinoma (ESCC). These findings prompted us to study, using nude mice, pathological roles of Rho proteins in human ESCC cells. Western blot analysis in ESCC cell lines, in addition to cell proliferation and in vitro migration assays, were performed to observe the malignant potential of RhoA and RhoC in untransfected and transfected cells. Constitutively active RhoA, RhoC and dominant negative RhoA (dnRhoA) proteins were transfected to ESCC (TE-1 and TE-2) cells. The stably transfected cells were injected into nude mice, and the growth and metastasis of these cells to the lungs were analysed. Tumour tissues were then examined using immunohistochemical methods for proteins Ki-67 (MIB-1), FAK, MMP-1, MMP-9 and TIMP-3. Protein levels of RhoA and RhoC in ESCC cell lines were visualised by Western blotting, and showed highest expression in TE-2 cells. Results from the migration assay illustrated that both RhoA and RhoC play a role in migration of ESCC cells. In TE-2 transfected cells, RhoC showed greater migration compared to RhoA. By using an experimental metastasis model in nude mice, RhoA was found to promote more tumour growth than RhoC, whereas RhoC induced lung metastasis in comparison to RhoA. Ki-67 labelling index was used to evaluate the proliferation potential of tumour tissue inoculated from nude mice. In TE-2 cells RhoA gave a proliferation capacity of 24.8±0.5, which was significantly higher than those of TE-2 RhoC 10±0.4 (P <0.01). Strong immunoreactivity for FAK, MMP-1 and MMP-9 proteins was present in all tumour cells. By contrast, loss of TIMP-3 expression was observed in all tumour cells. In conclusion, our results indicate that pro-oncogenic Rho proteins are involved in promoting tumour growth, cell migration and metastasis in human ESCC cells in nude mice. The results from this study suggest that active Rho proteins may induce a transforming effect that leads to a malignant phenotype. [Copyright &y& Elsevier]

Abstract: Paclitaxel, a potent anti-neoplastic agent, has been found to be effective against several tumours, including cervical cancer. However, the exact mechanism underlying the cytotoxic effects of paclitaxel, especially in the survival-signalling pathway, is poorly understood. The aim of this study was to investigate the molecular pathway of the cytotoxic effect of paclitaxel in human cervical cancer cell lines. Four human cervical cancer cell lines were treated for 24h with various concentration of paclitaxel, and the sensitivity was analysed by an MTT assay. The cell cycle progression and sub-G1 population were analysed by flow cytometry. Apoptosis was further measured by DNA fragmentation and microscope examination. The protein expression was determined by Western blot analysis. Our results showed that HeLa cells demonstrated the highest sensitivity to paclitaxel, whereas CaSki cells showed the lowest. In cervical cancer cells, paclitaxel induced apoptosis through an intrinsic pathway with prior G2/M arrest. In addition, we showed that paclitaxel downregulated the phosphorylation of Akt in both HeLa and CaSki cells. Interestingly, in CaSki cells, which were more suggestive of a resistant phenotype, paclitaxel induced the activation of mTOR as a downstream target of Akt. Pre-treatment with rapamycin inhibited activation of mTOR signalling and significantly enhanced the sensitivity of CaSki cells to paclitaxel by increasing apoptotic cell death. This effect was mediated, at least partly, through caspase activation. Overall, paclitaxel exerts its anti-tumour effects on cervical cancer cells by inducing apoptosis through intrinsic pathway, and rapamycin targeted to mTOR can sensitise paclitaxel-resistant cervical cancer cells. [Copyright &y& Elsevier]

Bacillus thuringiensis crystal proteins, well known to be toxic to certain insects but not pathogenic to mammals, are used as insecticidal proteins in agriculture and forest management. We here identified a crystal protein that is non-insecticidal and non-hemolytic but has strong cytocidal activity against various human cells with a markedly divergent target specificity, e.g. highly cytotoxic to HepG2 and Jurkat and less cytotoxic to the normal hepatocyte (HC) and HeLa. In slices of liver and colon cancer tissues, the toxin protein preferentially killed the cancer cells, leaving other cells unaffected. The cytocidal effect of the protein is non-apoptotic with swelling and fragmentation of the susceptible cells, although the apoptotic process does occur when the cell damage proceeded slowly. The amino acid sequence deduced from the nucleotide sequence of the cloned gene of the protein has little sequence homology with the insecticidal crystal proteins of B. thuringiensis. These observations raise the presence of a new group of the B. thuringiensis toxin and the possibility of new applications for the protein in the medical field. [ABSTRACT FROM AUTHOR]

The thermal responses of mouse colorectal carcinoma cells were investigated in the wild type cells and the transfected cells with human FUT1 gene which encodes α1,2fucosyltransferase. The heat sensitivity was observed to increase in the FUT1 gene transfected cells and the effect of hyperthermia at 44°C on these cells was demonstrated to be significant (P<0.001) to the wild type cells even though no remarkable difference in the expression of the heat shock protein, Hsp70 was found in these cells. Thus the expression of α1,2fucosylated antigens seemed to increase the heat sensitivity in mouse colorectal carcinoma cells. [Copyright &y& Elsevier]