Oncogenic MicroRNA-155 Down-regulates Tumor Suppressor CDC73 and Promotes Oral Squamous Cell Carcinoma Cell Proliferation IMPLICATIONS FOR CANCER THERAPEUTICS

The CDC73 gene is mutationally inactivated in hereditary and sporadic parathyroid tumors. It negatively regulates beta-catenin, cyclin D1, and c-MYC. Down-regulation of CDC73 has been reported in breast, renal, and gastric carcinomas. However, the reports regarding the role of CDC73 in oral squamous cell carcinoma (OSCC) are lacking. In this study we show that CDC73 is down-regulated in a majority of OSCC samples. We further show that oncogenic microRNA-155 (miR-155) negatively regulates CDC73 expression. Our experiments show that the dramatic up-regulation of miR-155 is an exclusive mechanism for down-regulation of CDC73 in a panel of human cell lines and a subset of OSCC patient samples in the absence of loss of heterozygosity, mutations, and promoter methylation. Ectopic expression of miR-155 in HEK293 cells dramatically reduced CDC73 levels, enhanced cell viability, and decreased apoptosis. Conversely, the delivery of a miR-155 antagonist (antagomir-155) to KB cells overexpressing miR-155 resulted in increased CDC73 levels, decreased cell viability, increased apoptosis, and marked regression of xenografts in nude mice. Cotransfection of miR-155 with CDC73 in HEK293 cells abrogated its pro-oncogenic effect. Reduced cell proliferation and increased apoptosis of KB cells were dependent on the presence or absence of the 3'-UTR in CDC73. In summary, knockdown of CDC73 expression due to overexpression of miR-155 not only adds a novelty to the list of mechanisms responsible for its down-regulation in different tumors, but the restoration of CDC73 levels by the use of antagomir-155 may also have an important role in therapeutic intervention of cancers, including OSCC.

Toll-Like Receptors 7, 8, and 9 Expression and Function in Primary Human Cervical Cancer Langerhans Cells Evidence of Anergy

Objective: Human papillomavirus oncoproteins E6 and E7 down modulate Toll-like receptor (TLR) 9 expression in infected keratinocytes. We explored the status of expression and function of TLR7, TLR8, and TLR9 in primary human Langerhans cells (LCs) isolated from cervical tumors. Methodology: Single-cell suspensions were made from fresh tissues of squamous cell carcinoma (International Federation of Gynecology and Obstetrics stage IB2); myeloid dendritic cells were purified using CD1c magnetic activated cell separation kits. Langerhans cells were further flow sorted into CD1a(+)CD207(+) cells. Acute monocytic leukemia cell line THP-1-derived LCs (moLCs) formed the controls. mRNA from flow-sorted LCs was reverse transcribed to cDNA and TLR7, TLR8, and TLR9 amplified. Monocyte-derived Langerhans cells and cervical tumor LCs were stimulated with TLR7, TLR8, and TLR9 ligands. Culture supernatants were assayed for interleukin (IL) 1 beta, IL-6, IL-10, IL-12p70, interferon (IFN) alpha, interferon gamma, and tumor necrosis factor (TNF) alpha by Luminex multiplex bead array. Human papillomavirus was genotyped. Results: We have for the first time demonstrated that the acute monocytic leukemia cell line THP-1 can be differentiated into LCs in vitro. Although these moLCs. expressed all the 3 TLRs, tumor LCs expressed TLR7 and TLR8, but uniformly lacked TLR9. Also, moLCs secreted IL-6, IL-1 beta, and tumor necrosis factor alpha to TLR8 ligand and interferon alpha in response to TLR9 ligand; in contrast, tumor LCs did not express any cytokine to any of the 3 TLR ligands. Human papillomavirus type 16 was one of the common human papillomavirus types in all cases. Conclusions: Cervical tumor LCs lacked TLR9 expression and were functionally anergic to all the 3: TLR7, TLR8, and TLR9 ligands, which may play a crucial role in immune tolerance. The exact location of block(s) in TLR7 and TLR8 signaling needs to be investigated, which would have important immunotherapeutic implications.

Antitumor and antioxidant status of Terminalia catappa against Ehrlich ascites carcinoma in Swiss albino mice

Objective: The present study was undertaken to evaluate the antitumor and antioxidant status of ethanol extract of Terminalia catappa leaves against Ehrlich ascites carcinoma (EAC) in Swiss albino mice. Materials and Methods: The leaves powder was extracted with Soxhlet apparatus and subjected to hot continuous percolation using ethanol (95% v/v). Tumor bearing animals was treated with 50 and 200 mg/kg of ethanol extract. EAC induced in mice by intraperitoneal injection of EAC cells 1 x 10(6) cells/mice. The study was assed using life span of EAC-bearing hosts, hematological parameters, volume of solid tumor mass and status of antioxidant enzymes such as lipid peroxidation (LPO), reduced glutathione (GSH), superoxide dismutase (SOD) and catalase (CAT) activities. Total phenolics and flavonoids contents from the leaves extract were also determined. Results: Total phenolics and flavonoids contents from the leaves extract were found 354.02 and 51.67 mg/g extract. Oral administration of ethanol extract of T. catappa (50 and 200 mg/kg) increased the life span (27.82% and 60.59%), increased peritoneal cell count (8.85 +/- 0.20 and 10.37 +/- 0.26) and significantly decreased solid tumor mass (1.16 +/- 0.14 cm(2)) at 200 mg/kg as compared with EAC-tumor bearing mice (P < 0.01). Hematological profile including red blood cell count, white blood cell count, hemoglobin (11.91 +/- 0.47 % g) and protein estimation were found to be nearly normal levels in extract-treated mice compared with tumor bearing control mice. Treatment with T. catappa significantly decreased levels of LPO and GSH, and increased levels of SOD and CAT activity (P < 0.01). Conclusion: T. catappa exhibited antitumor effect by modulating LPO and augmenting antioxidant defense systems in EAC bearing mice. The phenolic and flavonoid components in this extract may be responsible for antitumor activity.

Anticancer Effects of Brominated Indole Alkaloid Eudistomin H from Marine Ascidian Eudistoma viride Against Cervical Cancer Cells (HeLa)

Marine invertebrates called ascidians are prolific producers of bioactive substances. The ascidian Eudistoma viride, distributed along the Southeast coast of India, was investigated for its in vitro cytotoxic activity against human cervical carcinoma (HeLa) cells by the MTT assay. The crude methanolic extract of E. viride, with an IC50 of 53 mu g/ml, was dose-dependently cytotoxic. It was more potent at 100 mu g/ml than cyclohexamide (1 mu g/ml), reducing cell viability to 9.2%. Among nine fractions separated by chromatography, ECF-8 exhibited prominent cytoxic activity at 10 mu g/ml. The HPLC fraction EHF-21 of ECF-8 was remarkably dose- and time-dependently cytotoxic, with 39.8% viable cells at 1 mu g/ml compared to 51% in cyclohexamide-treated cells at the same concentration; the IC50 was 0.49,mu g/ml. Hoechst staining of HeLa cells treated with EHF-2I at 0.5 mu g/ml revealed apoptotic events such an cell shrinkage, membrane blebbing, chromatin condensation and formation of apoptotic bodies. Cell size and granularity study showed changes in light scatter, indicating the characteristic feature of cells dying by apoptosis. The cell-cycle analysis of HeLa cells treated with fraction EHF-21 at 1 mu g/ml showed the marked arrest of cells in G(0)/G(1), S and G(2)/M phases and an increase in the sub G(0)/G(1) population indicated an increase in the apoptotic cell population. The statistical analysis of the sub-G(1) region showed a dose-dependent induction of apoptosis. DNA fragmentation was also observed in HeLa cells treated with EHF-21. The active EHF-2I fraction, a brominated indole alkaloid Eudistomin H, led to apoptotic death of HeLa cells.

Genomic amplification upregulates estrogen-related receptor alpha and its depletion inhibits oral squamous cell carcinoma tumors in vivo

The ESRRA gene encodes a transcription factor and regulates several genes, such as WNT11 and OPN, involved in tumorigenesis. It is upregulated in several cancers, including OSCC. We have previously shown that the tumor suppressor miR-125a targets ESRRA, and its downregulation causes upregulation of ESRRA in OSCC. Upregulation of ESRRA in the absence of downregulation of miR-125a in a subset of OSCC samples suggests the involvement of an alternative mechanism. Using TaqMan (R) copy number assay, here we report for the first time that the genomic amplification of ESRRA causes its upregulation in a subset of OSCC samples. Ectopic overexpression of ESRRA led to accelerated cell proliferation, anchorage-independent cell growth and invasion, and inhibited apoptosis. Whereas, knockdown of ESRRA expression by siRNA led to reduced cell proliferation, anchorage-independent cell growth and invasion, and accelerated apoptosis. Furthermore, the delivery of a synthetic biostable ESRRA siRNA to OSCC cells resulted in regression of xenografts in nude mice. Thus, the genomic amplification of ESRRA is another novel mechanism for its upregulation in OSCC. Based on our in vitro and in vivo experiments, we suggest that targeting ESRRA by siRNA could be a novel therapeutic strategy for OSCC and other cancers.

Inhibition of p300 lysine acetyltransferase activity by luteolin reduces tumor growth in head and neck squamous cell carcinoma (HNSCC) xenograft mouse model

Chromatin acetylation is attributed with distinct functional relevance with respect to gene expression in normal and diseased conditions thereby leading to a topical interest in the concept of epigenetic modulators and therapy. We report here the identification and characterization of the acetylation inhibitory potential of an important dietary flavonoid, luteolin. Luteolin was found to inhibit p300 acetyltransferase with competitive binding to the acetyl CoA binding site. Luteolin treatment in a xenografted tumor model of head and neck squamous cell carcinoma (HNSCC), led to a dramatic reduction in tumor growth within 4 weeks corresponding to a decrease in histone acetylation. Cells treated with luteolin exhibit cell cycle arrest and decreased cell migration. Luteolin treatment led to an alteration in gene expression and miRNA profile including up-regulation of p53 induced miR-195/215, let7C; potentially translating into a tumor suppressor function. It also led to down regulation of oncomiRNAs such as miR-135a, thereby reflecting global changes in the microRNA network. Furthermore, a direct correlation between the inhibition of histone acetylation and gene expression was established using chromatin immunoprecipitation on promoters of differentially expressed genes. A network of dysregulated genes and miRNAs was mapped along with the gene ontology categories, and the effects of luteolin were observed to be potentially at multiple levels: at the level of gene expression, miRNA expression and miRNA processing.