ETHNOPHARMACOLOGICAL RELEVANCE: Amorphophalli Rhizoma has been widely used as an adjuvant treatment for advanced or metastatic breast cancer, pancreatic cancer, hepatoma, and malignant lymphoma, but its molecular mechanism of action for treatment of metastatic triple-negative breast cancer (TNBC) is generally poorly understood.
AIM OF THE STUDY: To investigate genomic changes related to the inhibitory effect of Amorphophalli Rhizoma and to elucidate the molecular mechanism of this inhibition in MDA-MB-231 TNBC cells.
MATERIALS AND METHODS: Gene chip analysis was employed to explore genomic changes caused by Amorphophalli Rhizoma in TNBC cells. Potential classical signaling pathways, upstream regulators, functions, regulatory effects and gene interaction networks were analyzed by Ingenuity Pathway Analysis (IPA). Real-time quantitative PCR (RT-qPCR) and RNA interference (RNAi) assays were used to clarify the roles of potential target genes.
RESULTS: In total, 536 significantly upregulated and 648 significantly downregulated genes were identified between the group treated with Amorphophalli Rhizoma extract and that treated with vehicle. Many of these differentially expressed genes (DEGs) in TNBC cells are involved in DNA replication, recombination and repair, the cell cycle, and cellular assembly and organization. Attenuation of KNL1, OLFML2A, RTKN2 and SGO1 gene expression by Amorphophalli Rhizoma significantly induced cell cycle arrest and suppressed cell proliferation and migration.
CONCLUSIONS: The inhibitory effects of Amorphophalli Rhizoma in TNBC cells likely occur through regulation of the spindle checkpoint, chromosomal and centrosomal instability, and cell membrane stability.

BACKGROUND: Emerging evidence indicate that miRNAs play an important role on gastric cancer (GC) progression via regulating several downstream targets, but it is still partially uncovered. This study aimed to explore the molecular mechanisms of GC by comprehensive analysis of mRNAs and miRNA expression profiles.
METHODS: The mRNA and miRNA expression profiles of GSE79973 and GSE67354 downloaded from Gene Expression Omnibus were used to analyze the differentially expressed genes (DEGs) and DE-miRNAs among GC tissues and normal tissues. Then, targets genes of DE-miRNAs were predicted and the DE-miRNA-DEG regulatory network was constructed. Next, function enrichment analysis of the overlapped genes between the predicted DE-miRNAs targets and DEGs was performed and a protein-protein interactions network of overlapped genes was constructed. Finally, RT-PCR analysis was performed to detect the expression levels of several key DEGs and DE-miRNAs.
RESULTS: A set of 703 upregulated and 600 downregulated DEGs, as well as 8 upregulated DE-miRNAs and 27 downregulated DE-miRNAs were identified in GC tissue. hsa-miR-193b-3p and hsa-miR-148a-3p, which targeted most DEGs, were highlighted in the DE-miRNA-DEG regulatory network, as well as hsa-miR-1179, which targeted KNL1, was newly predicted to be associated with GC. In addition, NCAPG, which is targeted by miR-193b-3p, and KNL1, which is targeted by hsa-miR-1179, had higher degrees in the PPI network. RT-qPCR results showed that hsa-miR-148a-3p, hsa-miR-193b-3p, and hsa-miR-1179 were downregulated, and NCAPG and KNL1 were upregulated in GC tissues; this is consistent with our bioinformatics-predicted results.
CONCLUSIONS: The downregulation of miR-193b-3p might contribute to GC cell proliferation by mediating the upregulation of NCAPG; as additionally, the downregulation of miR-193b-3p might contribute to the mitotic nuclear division of GC cells by mediating the upregulation of KNL1.

Here we present the analysis of alternative splicing events on an example of glioblastoma cell culture samples using a set of computer tools in combination with database integration. The gene expression profiles of glioblastoma were obtained from cell culture samples of primary glioblastoma which were isolated and processed for RNA extraction. Transcriptome profiling of normal brain samples and glioblastoma were done by Illumina sequencing. The significant differentially expressed exon-level probes and their corresponding genes were identified using a combination of the splicing index method. Previous studies indicated that tumor-specific alternative splicing is important in the regulation of gene expression and corresponding protein functions during cancer development. Multiple alternative splicing transcripts have been identified as progression markers, including generalized splicing abnormalities and tumor- and stage-specific events. We used a set of computer tools which were recently applied to analysis of gene expression in laboratory animals to study differential splicing events. We found 69 transcripts that are differentially alternatively spliced. Three cancer-associated genes were considered in detail, in particular: APP (amyloid beta precursor protein), CASC4 (cancer susceptibility candidate 4) and TP53. Such alternative splicing opens new perspectives for cancer research.

The Cancer Genome Atlas (TCGA) offers an unprecedented opportunity to identify small-molecule binding sites on proteins with overexpressed mRNA levels that correlate with poor survival. Here, we analyze RNA-seq and clinical data for 10 tumor types to identify genes that are both overexpressed and correlate with patient survival. Protein products of these genes were scanned for binding sites that possess shape and physicochemical properties that can accommodate small-molecule probes or therapeutic agents (druggable). These binding sites were classified as enzyme active sites (ENZ), protein-protein interaction sites (PPI), or other sites whose function is unknown (OTH). Interestingly, the overwhelming majority of binding sites were classified as OTH. We find that ENZ, PPI, and OTH binding sites often occurred on the same structure suggesting that many of these OTH cavities can be used for allosteric modulation of enzyme activity or protein-protein interactions with small molecules. We discovered several ENZ (PYCR1, QPRT, and HSPA6) and PPI (CASC5, ZBTB32, and CSAD) binding sites on proteins that have been seldom explored in cancer. We also found proteins that have been extensively studied in cancer that have not been previously explored with small molecules that harbor ENZ (PKMYT1, STEAP3, and NNMT) and PPI (HNF4A, MEF2B, and CBX2) binding sites. All binding sites were classified by the signaling pathways to which the protein that harbors them belongs using KEGG. In addition, binding sites were mapped onto structural protein-protein interaction networks to identify promising sites for drug discovery. Finally, we identify pockets that harbor missense mutations previously identified from analysis of TCGA data. The occurrence of mutations in these binding sites provides new opportunities to develop small-molecule probes to explore their function in cancer.

The D40 gene encodes a kinetochore protein that plays an essential role in kinetochore formation during mitosis. Short inhibitory RNA against D40, D40 siRNA, has been shown to deplete the D40 protein in the human cancer cell line HeLa, which harbors wild-type p53, and this activity was followed by the significant inhibition of cell growth and induction of apoptotic cell death. The p53-null cancer cell line, PC-3M-luc, is also sensitive to the significant growth inhibition and cell death induced by D40 siRNA. The growth of PC-3M-luc tumors transplanted into nude mice was inhibited by the systemic administration of D40 siRNA and the atelocollagen complex. Furthermore, D40 siRNA significantly inhibited growth and induced apoptotic cell death in a cell line with a gain-of-function (GOF) mutation in p53, MDA-MB231-luc, and also inhibited the growth of tumors transplanted into mice when administered as a D40 siRNA/atelocollagen complex. These results indicated that D40 siRNA induced apoptotic cell death in human cancer cell lines, and inhibited their growth in vitro and in vivo regardless of p53 status. Therefore, D40 siRNA is a potential candidate anti-cancer reagent.

Glioblastoma multiforme (GBM) is one of the most malignant and aggressive tumors, and has a very poor prognosis with a mean survival time of <2 years, despite intensive treatment using chemo-radiation. Therefore, novel therapeutic approaches including immunotherapy have been developed against GBM. For the purpose of identifying novel target antigens contributing to GBM treatment, we developed 17 primary glioma cell lines derived from high-grade glioma patients, and analyzed the expression of various tumor antigens and glioma-associated markers using a quantitative PCR and immunohistochemistry (IHC). A quantitative PCR using 54 cancer-testis (CT) antigen-specific primers showed that 36 CT antigens were positive in at least 1 of 17 serum-derived cell lines, and 17 antigens were positive in >50% cell lines. Impressively, 6 genes (BAGE, MAGE-A12, CASC5, CTAGE1, DDX43 and IL-13RA2) were detected in all cell lines. The expression of other 13 glioma-associated antigens than CT genes were also investigated, and 10 genes were detected in >70% cell lines. The expression of CT antigen and glioma-associated antigen genes with a high frequency were also verified in IHC analysis. Moreover, a relationship of antigen gene expressions with a high frequency to overall survival was investigated using the Repository of Molecular Brain Neoplasia Data (REMBRANDT) database of the National Cancer Institute, and expression of 6 genes including IL-13RA2 was inversely correlated to overall survival time. Furthermore, 4 genes including DDX43, TDRD1, HER2 and gp100 were identified as MGMT-relevant factors. In the present study, several CT antigen including novel genes were detected in high-grade glioma primary cell lines, which might contribute to developing novel immunotherapy and glioma-specific biomarkers in future.

Cancer/testis (CT) antigens encoded by CT genes are immunogenic antigens, and the expression of CT gene is strictly restricted to only the testis among mature organs. Therefore, CT antigens are promising candidates for cancer immunotherapy. In a previous study, we identified a novel CT antigen, DNAJB8. DNAJB8 was found to be preferentially expressed in cancer stem-like cells (CSCs)/cancer-initiating cells (CICs), and it is thus a novel CSC antigen. In this study, we hypothesized that CT genes are preferentially expressed in CSCs/CICs rather than in non-CSCs/-CICs and we examined the expression of CT genes in CSCs/CICs. The expression of 74 CT genes was evaluated in side population (SP) cells (=CSC) and main population (MP) cells (=non-CSC) derived from LHK2 lung adenocarcinoma cells, SW480 colon adenocarcinoma cells and MCF7 breast adenocarcinoma cells by RT-PCR and real-time PCR. Eighteen genes (MAGEA2, MAGEA3, MAGEA4, MAGEA6, MAGEA12, MAGEB2, GAGE1, GAGE8, SPANXA1, SPANXB1, SPANXC, XAGE2, SPA17, BORIS, PLU-1, SGY-1, TEX15 and CT45A1) showed higher expression levels in SP cells than in MP cells, whereas 10 genes (BAGE1, BAGE2, BAGE4, BAGE5, XAGE1, LIP1, D40, HCA661, TDRD1 and TPTE) showed similar expression levels in SP cells and MP cells. Thus, considerable numbers of CT genes showed preferential expression in CSCs/CICs. We therefore propose a novel sub-category of CT genes in this report: cancer/testis/stem (CTS) genes.

UNLABELLED: Epigenetic regulation of genes involves the coordination of DNA methylation and histone modifications to maintain transcriptional status. These two features are frequently disrupted in malignancy such that critical genes succumb to inactivation. 5-aza-2'-deoxycytidine (5-aza-dC) is an agent which inhibits DNA methyltransferase, and holds great potential as a treatment for cancer, yet the extent of its effectiveness varies greatly between tumour types. Previous evidence suggests expression status after 5-aza-dC exposure cannot be explained by the DNA methylation status alone.
AIM: We sought to identify chromatin changes involved with short and long term gene reactivation following 5-aza-dC exposure. Two colorectal cancer cell lines, HCT116 and SW480, were treated with 5-aza-dC and then grown in drug-free media to allow DNA re-methylation. DNA methylation and chromatin modifications were assessed with bisulfite sequencing and Chromatin Immuno-Precipitation analysis.
RESULTS: Increased H3 acetylation, H3K4 tri-methylation and loss of H3K27 tri-methylation were associated with reactivation. Hypermethylated genes that did not show increased acetylation were transiently expressed with 5-aza-dC treatment before reverting to an inactive state. Three reactivated genes, CDO1, HSPC105 and MAGEA3, were still expressed 10 days post 5-aza-dC treatment and displayed localised hypomethylation at the transcriptional start site, and also an increased enrichment of histone H3 acetylation.
CONCLUSIONS: These observations suggest that hypomethylation alone is insufficient to reactivate silenced genes and that increased Histone H3 acetylation in unison with localised hypomethylation allows long term reversion of these epigenetically silenced genes. This study suggests that combined DNA methyltransferase and histone deacetylase inhibitors may aid long term reactivation of silenced genes.

We have previously identified and cloned a human gene, D40, that is preferentially expressed in testis among normal organs, while it is widely expressed in various human tumor cell lines and primary tumors derived from different organs. In this report, we have examined the expression and localization of this protein in human testis with an antibody specific to D40 protein. In Western analyses, the anti-D40 antibody recognized a major band with a molecular mass of 300 kDa and a minor band of 250 kDa. These bands were not observed in the testis lysates from patients with Sertoli-cell-only syndrome and with Kleinfelter syndrome, who lack germ cells of the testis, indicating that D40 protein is expressed in the germ cells of normal testis. Immunohistochemical studies have revealed that D40 protein is highly expressed in spermatocytes and in the pre-acrosome of round spermatids. In the acrosome, D40 protein expression is observed not inside but outside the acrosome membrane. This is consistent with the finding that the amino-acid sequence at the amino terminal of the D40 protein lacks a hydrophobic signal peptide that is required for proteins to translocate to the membrane. Expression of D40 protein is observed in the acrosome of ejaculated spermatozoa as well, although the level is low compared with that in the pre-acrosome of spermatids. These results suggest that D40 protein plays important roles in spermatogenesis, especially in the formation and maintenance of the acrosome.

Translocations interrupting the mixed lineage leukemia gene (MLL) occur in 7-10% of acute lymphoblastic leukemia (ALL) and 5-6% of acute myeloid leukemia (AML) cases. One of these translocations, t(11;15)(q23;q14), occurs rarely in both ALL and AML. The gene on chromosome 15, AF15q14, was cloned recently in a patient with AML-M4. We have identified the same gene in a de novo T-ALL patient. However, both the MLL and AF15q14 breakpoints in these patients differed: in the previously reported AML-M4, both gene breaks were within exons, while in our ALL case the MLL break is intronic and the AF15q14 break is exonic. The MLL-AF15q14 fusion described previously shares no AF15q14 residues in common with the chimera reported here. The fusion proteins also differ with respect to MLL--the previously described fusion contains 55 extra amino acids as its MLL break is in exon 11, while the chimera we report breaks in intron 9. Contrary to the originally described normal AF15q14 (5925-bp cDNA encoding a 1833-aa protein), we identify a 7542-bp cDNA and a 2342-aa AF15q14 protein. AF15q14 appears identical to an mRNA previously found to be expressed in melanoma rendered nontumorigenic by microcell-mediated introduction of normal chromosome 6, suggesting the gene may function normally to suppress cell growth and/or enhance maturation.

The mixed lineage leukemia gene (MLL, also known as HRX, ALL-1 and Htrx) located at 11q23 is involved in translocations with over 40 different chromosomal bands in a variety of leukemia subtypes. Here we report our analysis of a rare but recurring translocation, t(11;15)(q23;q14). This translocation has been described in a small subset of cases with both acute myeloblastic leukemia and ALL. Recent studies have shown that MLL is fused to AF15q14 in the t(11;15). Here we analyse a sample from another patient with this translocation and confirm the presence of an MLL-AF15q14 fusion. However, we have also identified and cloned another fusion transcript from the same patient sample. In this fusion transcript, MLL is fused to a novel gene, MLL partner containing FYVE domain (MPFYVE). Both MLL-AF15q14 and MLL-MPFYVE are in-frame fusion transcripts with the potential to code for novel fusion proteins. MPFYVE is also located on chromosome 15, approximately 170 kb telomeric to AF15q14. MPFYVE contains a highly conserved motif, the FYVE domain which, in other proteins, has been shown to bind to phosphotidyl-inositol-3 phosphate (PtdIns(3)P). The MLL-MPFYVE fusion may be functionally important in the leukemia process in at least some patients containing this translocation.

We found a significant correlation between lung cancer in smokers and the expression of a human gene, D40, predominantly expressed in testis and cancers. In an attempt to clone a novel human gene, we screened a cDNA library derived from a human B cell line and obtained a cDNA clone that we refer to as D40. A search for public databases for sequence homologies showed that the D40 gene is identical to AF15q14. D40 mRNA is predominantly expressed in normal testis tissue. However, this gene is also expressed in various human tumour cell lines and primary tumours derived from various organs and tissues, such as lung cancer. We examined the relationship between D40 expression and clinico-pathological characteristics of tumours in primary lung cancer. D40 expression did not significantly correlate with either histological type or pathological tumour stage. However, D40 expression was observed more frequently in poorly differentiated tumours than in well or moderately differentiated ones. Furthermore, the incidence of D40 expression was significantly higher in tumours from patients who smoke than in those from non-smokers. D40/AF15q14 is the first gene in the cancer/testis family for which expression is related to the smoking habits of cancer patients.

In haematopoietic malignancies the MLL gene, located on chromosome 11q23, is frequently disrupted by chromosome rearrangement, generally resulting in fusion to various partner genes. We have previously reported a t(11;15)(q23;q14) in a case of acute myeloblastic leukaemia. Here, we report the cloning of a novel MLL partner, AF15q14, at chromosome 15q14. In this translocation, the breakpoint occurred in exon 8 of MLL and exon 10 of AF15q14. The normal AF15q14 transcripts of approximately 8.5 kb in size, are expressed in different tumoral cell lines, in a variety of normal tissues, and in all the foetal tissues tested. Sequencing of AF15q14 cDNA revealed a putative open reading frame of 1833 amino acids that had no homology with any other known protein. The C-terminal end of the putative AF15q14 contained a bipartite nuclear localization site. The translocation t(11;15) preserved the open reading frame between MLL and the 3' end of AF15q14. The contribution of AF15q14 to the fusion protein was only 85 amino acids. Immunofluorescence staining experiments with expression vectors encoding these 85 amino acids confirmed the functionality of the predicted nuclear localization site.

A major obstacle for the effective treatment of cancer is the phenomenon of multidrug resistance (MDR) exhibited by many tumor cells. Many, but not all, MDR cells exhibit membrane-associated P-glycoprotein (P-gp), a drug efflux pump. However, most mechanisms of MDR are complex, employing P-gp in combination with other, ill-defined activities. Altered cytosolic pH (pHi) has been implicated to play a role in drug resistance. In the current study, we investigated mechanisms of pHi regulation in drug-sensitive (MCF-7/S) and drug-resistant human breast cancer cells. Of the drug-resistant lines, one contained P-gp (MCF-7/DOX; also referred to as MCF-7/D40) and one did not (MCF-7/MITOX). The resting steady-state pHi was similar in the three cell lines. In addition, in all the cell lines, HCO3- slightly acidified pHi and increased the rates of pHi recovery after an acid load, indicating the presence of anion exchanger (AE) activity. These data indicate that neither Na+/H+ exchange nor AE is differentially expressed in these cell lines. The presence of plasma membrane vacuolar-type H+-ATPase (pmV-ATPase) activity in these cell lines was then investigated. In the absence of Na+ and HCO3-, MCF-7/S cells did not recover from acid loads, whereas MCF-7/MITOX and MCF-7/DOX cells did. Furthermore, recovery of pHi was inhibited by bafilomycin A1 and NBD-Cl, potent V-ATPase inhibitors. Attempts to localize V-ATPase immunocytochemically at the plasma membranes of these cells were unsuccessful, indicating that V-ATPase is not statically resident at the plasma membrane. Consistent with this was the observation that release of endosomally trapped dextran was more rapid in the drug-resistant, compared with the drug-sensitive cells. Furthermore, the drug-resistant cells entrapped doxorubicin into intracellular vesicles whereas the drug-sensitive cells did not. Hence, it is hypothesized that the measured pmV-ATPase activity in the drug-resistant cells is a consequence of rapid endomembrane turnover. The potential impact of this behavior on drug resistance is examined in a companion manuscript.

The effect of isoflavones on the growth of the human breast carcinoma cell lines, MDA-468 (estrogen receptor negative), and MCF-7 and MCF-7-D-40 (estrogen receptor positive), has been examined. Genistein is a potent inhibitor of the growth of each cell line (IC50 values from 6.5 to 12.0 micrograms/ml), whereas biochanin A and daidzein are weaker growth inhibitors (IC50 values from 20 to 34 micrograms/ml). The isoflavone beta-glucosides, genistin and daidzin, have little effect on growth (IC50 values greater than 100 micrograms/ml). The presence of the estrogen receptor is not required for the isoflavones to inhibit tumor cell growth (MDA-468 vs MCF-7 cells). In addition, the effects of genistein and biochanin A are not attenuated by overexpression of the multi-drug resistance gene product (MCF-7-D40 vs MCF-7 cells).