Detection of EBV Infection and Gene Expression in Oral Cancer from Patients in Taiwan by Microarray Analysis

Epstein-Barr virus is known to cause nasopharyngeal carcinoma. Although oral cavity is located close to the nasal pharynx, the pathogenetic role of Epstein-Barr virus (EBV) in oral cancers is unclear. This molecular epidemiology study uses EBV genomic microarray (EBV-chip) to simultaneously detect the prevalent rate and viral gene expression patterns in 57 oral squamous cell carcinoma biopsies (OSCC) collected from patients in Taiwan. The majority of the specimens (82.5%) were EBV-positive that probably expressed coincidently the genes for EBNAs, LMP2A and 2B, and certain structural proteins. Importantly, the genes fabricated at the spots 61 (BBRF1, BBRF2, and BBRF3) and 68 (BDLF4 and BDRF1) on EBV-chip were actively expressed in a significantly greater number of OSCC exhibiting exophytic morphology or ulceration than those tissues with deep invasive lesions (P = .0265 and .0141, resp.). The results may thus provide the lead information for understanding the role of EBV in oral cancer pathogenesis

Tailor-Made Zinc-Finger Transcription Factors Activate FLO11 Gene Expression with Phenotypic Consequences in the Yeast Saccharomyces cerevisiae

Cys2His2 zinc fingers are eukaryotic DNA-binding motifs, capable of distinguishing different DNA sequences, and are suitable for engineering artificial transcription factors. In this work, we used the budding yeast Saccharomyces cerevisiae to study the ability of tailor-made zinc finger proteins to activate the expression of the FLO11 gene, with phenotypic consequences. Two three-finger peptides were identified, recognizing sites from the 5′ UTR of the FLO11 gene with nanomolar DNA-binding affinity. The three-finger domains and their combined six-finger motif, recognizing an 18-bp site, were fused to the activation domain of VP16 or VP64. These transcription factor constructs retained their DNA-binding ability, with the six-finger ones being the highest in affinity. However, when expressed in haploid yeast cells, only one three-finger recombinant transcription factor was able to activate the expression of FLO11 efficiently. Unlike in the wild-type, cells with such transcriptional activation displayed invasive growth and biofilm formation, without any requirement for glucose depletion. The VP16 and VP64 domains appeared to act equally well in the activation of FLO11 expression, with comparable effects in phenotypic alteration. We conclude that the functional activity of tailor-made transcription factors in cells is not easily predicted by the in vitro DNA-binding activity

A Synthetic Podophyllotoxin Derivative Exerts Anti-Cancer Effects by Inducing Mitotic Arrest and Pro-Apoptotic ER Stress in Lung Cancer Preclinical Models

<div><p>Some potent chemotherapy drugs including tubulin-binding agents had been developed from nature plants, such as podophyllotoxin and paclitaxel. However, poor cytotoxic selectivity, serious side-effects, and limited effectiveness are still the major concerns in their therapeutic application. We developed a fully synthetic podophyllotoxin derivative named Ching001 and investigated its anti-tumor growth effects and mechanisms in lung cancer preclinical models. Ching001 showed a selective cytotoxicity to different lung cancer cell lines but not to normal lung cells. Ching001 inhibited the polymerization of microtubule resulting in mitotic arrest as evident by the accumulation of mitosis-related proteins, survivin and aurora B, thereby leading to DNA damage and apoptosis. Ching001 also activated pro-apoptotic ER stress signaling pathway. Intraperitoneal injection of 2 mg/kg Ching001 significantly inhibited the tumor growth of A549 xenograft, while injection of 0.2 mg/kg Ching001 decreased the lung colonization ability of A549 cells in experimental metastasis assay. These anti-tumor growth and lung colonization inhibition effects were stronger than those of paclitaxel treatment at the same dosage. The xenograft tumor tissue stains further confirmed that Ching001 induced mitosis arrest and tumor apoptosis. In addition, the hematology and biochemistry tests of blood samples as well as tissue examinations indicated that Ching001 treatment did not show apparent organ toxicities in tested animals. We provided preclinical evidence that novel synthetic microtubule inhibitor Ching001, which can trigger DNA damage and apoptosis by inducing mitotic arrest and ER stress, is a potential anti-cancer compound for further drug development.</p></div

Ching001 induces M-phase arrest and DNA damage.

<p>(<b>A</b>) Western blot analyses of mitosis-related proteins after 1 µM Ching001 treatment for indicated times in lung cancer cell lines. (<b>B</b>) Dysregulation of M-phase cell cycle induced by Ching001. Lung cancer cell lines were analyzed with survivin (green), aurora B (red), and DAPI (blue) after 1 µM Ching001 treatment for 24 h. DMSO was used as solvent control. Scale bars: 30 µm.</p

Ching001 inhibits A549 xenograft growth via M-phase arrest and apoptosis.

<p>(<b>A</b>) ICR-nude mice bearing the established A549 tumors (∼50 mm³) were treated with Ching001 (0.4 mg/kg or 2 mg/kg) via intraperitoneal injection on day1, day3, day5, day7, and day9 (as indicated by arrow heads). A known microtubule inhibitor, paclitaxel (4 mg/kg), was used for comparison. The tumor volumes (left) and body weight (right) were measured on every other day till day35. Points, mean; bars, ±SEM. *: <i>P</i><0.05, **: <i>P</i><0.01, ***: <i>P</i><0.001. (<b>B</b>) The activated caspase-3 IHC staining of the tumor tissue of ICR-nude mice taken from solvent control group and Ching001 treatment (2 mg/kg) group. Original magnification×200. (<b>C</b>) The tissue immunofluorescence of survivin (green), aurora B (red), and DAPI (blue) of tumor xenograft from solvent control mice and Ching001 treated mice (2 mg/kg). The arrows indicated the mitotic cells in solvent control tumor. The enlarged figure represents aberrant chromosomes after Ching001 treatment. Scale bars: 30 µm.</p