Inhibition of Chemoresistance in Primary Tumor Cells by Camellia sinensis non fermentatum Extract Noviphenone (NPE®).

BACKGROUND/AIM: Despite improvements in cancer therapy, life expectancy after tumor recurrence remains low. Relapsed cancer is characterized by drug resistance, often mediated through overexpression of multidrug resistance (MDR) genes. Camellia sinensis non fermentatum extract is known for its anticancer properties in several cancer cell lines and might improve cancer therapy outcome after tumor recurrence. MATERIALS AND METHODS: Embryonal rhabdomyosarcoma cell lines, alveolar rhabdomyosarcoma cell lines and primary rhabdomyosarcoma MAST139 cells were used to test NPE® effects on cell viability in combination with chemotherapeutic agents. Cell viability was measured by the WST-1 assay and CV staining. Gene expression levels of chemotherapy-induced efflux pumps and their activity was assessed upon NPE® treatment by measuring doxorubicin retention through evaluation of the autofluorescence signal. RESULTS: Administration of increasing doxorubicin concentrations triggered immediate adaptation to the drug, which was surprisingly overcome by the addition of NPE®. Investigating the mechanism of immediate adaptation, MDR1 gene overexpression was observed upon doxorubicin treatment. Although NPE® did not alter pump gene expression, it was able to reduce pump activity, thus allowing the chemotherapeutic agent to stay inside the cells to exert its full anticancer activity. CONCLUSION: NPE® might improve chemotherapeutic treatment by re-sensitizing relapsed tumors to anticancer drugs. Fighting MDR represents the key to overcome tumor relapse and improve the overall survival of cancer patients.

Characterization of Dnmt1 Binding and DNA Methylation on Nucleosomes and Nucleosomal Arrays.

The packaging of DNA into nucleosomes and the organisation into higher order structures of chromatin limits the access of sequence specific DNA binding factors to DNA. In cells, DNA methylation is preferentially occuring in the linker region of nucleosomes, suggesting a structural impact of chromatin on DNA methylation. These observations raise the question whether DNA methyltransferases are capable to recognize the nucleosomal substrates and to modify the packaged DNA. Here, we performed a detailed analysis of nucleosome binding and nucleosomal DNA methylation by the maintenance DNA methyltransferase Dnmt1. Our binding studies show that Dnmt1 has a DNA length sensing activity, binding cooperatively to DNA, and requiring a minimal DNA length of 20 bp. Dnmt1 needs linker DNA to bind to nucleosomes and most efficiently recognizes nucleosomes with symmetric DNA linkers. Footprinting experiments reveal that Dnmt1 binds to both DNA linkers exiting the nucleosome core. The binding pattern correlates with the efficient methylation of DNA linkers. However, the enzyme lacks the ability to methylate nucleosomal CpG sites on mononucleosomes and nucleosomal arrays, unless chromatin remodeling enzymes create a dynamic chromatin state. In addition, our results show that Dnmt1 functionally interacts with specific chromatin remodeling enzymes to enable complete methylation of hemi-methylated DNA in chromatin.

PLK1 phosphorylates PAX3-FOXO1, the inhibition of which triggers regression of alveolar Rhabdomyosarcoma.

Pediatric tumors harbor very low numbers of somatic mutations and therefore offer few targets to improve therapeutic management with targeted drugs. In particular, outcomes remain dismal for patients with metastatic alveolar rhabdomyosarcoma (aRMS), where the chimeric transcription factor PAX3/7-FOXO1 has been implicated but problematic to target. In this report, we addressed this challenge by developing a two-armed screen for druggable upstream regulatory kinases in the PAX3/7-FOXO1 pathway. Screening libraries of kinome siRNA and small molecules, we defined PLK1 as an upstream-acting regulator. Mechanistically, PLK1 interacted with and phosphorylated PAX3-FOXO1 at the novel site S503, leading to protein stabilization. Notably, PLK1 inhibition led to elevated ubiquitination and rapid proteasomal degradation of the PAX3-FOXO1 chimeric oncoprotein. On this basis, we embarked on a preclinical validation of PLK1 as a target in a xenograft mouse model of aRMS, where the PLK1 inhibitor BI 2536 reduced PAX3-FOXO1-mediated gene expression and elicited tumor regression. Clinically, analysis of human aRMS tumor biopsies documented high PLK1 expression to offer prognostic significance for both event-free survival and overall survival. Taken together, these preclinical studies validate the PLK1-PAX3-FOXO1 axis as a rational target to treat aRMS.

Small-molecule screen identifies modulators of EWS/FLI1 target gene expression and cell survival in Ewing's sarcoma.

Ewing's sarcoma family of tumors (EFT) is characterized by the presence of chromosomal translocations leading to the expression of oncogenic transcription factors such as, in the majority of cases, EWS/FLI1. Because of its key role in Ewing's sarcoma development and maintenance, EWS/FLI1 represents an attractive therapeutic target. Here, we characterize PHLDA1 as a novel direct target gene whose expression is repressed by EWS/FLI1. Using this gene and additional specific well-characterized target genes such as NROB1, NKX2.2 and CAV1, all activated by EWS/FLI1, as a read-out system, we screened a small-molecule compound library enriched for FDA-approved drugs that modulated the expression of EWS/FLI1 target genes. Among a hit-list of nine well-known drugs such as camptothecin, fenretinide, etoposide and doxorubicin, we also identified the kinase inhibitor midostaurin (PKC412). Subsequent experiments demonstrated that midostaurin is able to induce apoptosis in a panel of six Ewing's sarcoma cell lines in vitro and can significantly suppress xenograft tumor growth in vivo. These results suggest that midostaurin might be a novel drug that is active against Ewing's cells, which might act by modulating the expression of EWS/FLI1 target genes.

The USP7/Dnmt1 complex stimulates the DNA methylation activity of Dnmt1 and regulates the stability of UHRF1.

Aberrant DNA methylation is often associated with cancer and the formation of tumors; however, the underlying mechanisms, in particular the recruitment and regulation of DNA methyltransferases remain largely unknown. In this study, we identified USP7 as an interaction partner of Dnmt1 and UHRF1 in vivo. Dnmt1 and USP7 formed a soluble dimer complex that associated with UHRF1 as a trimeric complex on chromatin. Complex interactions were mediated by the C-terminal domain of USP7 with the TS-domain of Dnmt1, whereas the TRAF-domain of USP7 bound to the SRA-domain of UHRF1. USP7 was capable of targeting UHRF1 for deubiquitination and affects UHRF1 protein stability in vivo. Furthermore, Dnmt1, UHRF1 and USP7 co-localized on silenced, methylated genes in vivo. Strikingly, when analyzing the impact of UHRF1 and USP7 on Dnmt1-dependent DNA methylation, we found that USP7 stimulated both the maintenance and de novo DNA methylation activity of Dnmt1 in vitro. Therefore, we propose a dual role of USP7, regulating the protein turnover of UHRF1 and stimulating the enzymatic activity of Dnmt1 in vitro and in vivo.

Regulation of mitochondrial polarity in mouse and human oocytes: the influence of cumulus derived nitric oxide.

Whether exogenous factors influenced the level of mitochondrial polarity (DeltaPsim) in the subplasmalemmal cytoplasm of the oocyte was investigated with denuded and cumulus-enclosed human and mouse oocytes between the germinal vesicle and metaphase II stage. Co-culture of denuded oocytes with cumulus masses or primary cumulus cell cultures demonstrated a 'proximity' effect with respect to the detectable level of DeltaPsim in the oocyte. The specificity and reversibility of this effect on subplasmalemmal mitochondria were shown by repeated repositioning between cellular and acellular regions, which sequentially down- or up-regulated DeltaPsim. Experimental studies with a nitric oxide (NO) donor and inhibitor of NO synthase indicate that NO produced by cumulus cells has a regulatory influence on DeltaPsim in the subplasmalemmal cytoplasm of the corresponding oocyte. Culture of denuded and cumulus-enclosed (intact) oocytes in low and high oxygen atmospheres suggests that competition between oxygen and NO at the mitochondrial level may regulate the level of DeltaPsim and maintain mitochondria homeostasis in the pre-ovulatory oocyte, with a shift to higher polarity occurring after ovulation. The role of exogenous influences on oocyte DeltaPsim is discussed with respect to the regulation of developmental processes in the oocyte and early embryo.