Ets2 maintains hTERT gene expression and breast cancer cell proliferation by interacting with c-Myc

Human telomerase reverse transcriptase (hTERT) underlies cancer cell immortalization, and the expression of hTERT is regulated strictly at the gene transcription. Here, we report that transcription factor Ets2 is required for hTERT gene expression and breast cancer cell proliferation. Silencing Ets2 induces a decrease of hTERT gene expression and increase in human breast cancer cell death. Reconstitution with recombinant hTERT rescues the apoptosis induced by Ets2 depression. In vitro and in vivo analyses show that Ets2 binds to the EtsA and EtsB DNA motifs on the hTERT gene promoter. Mutation of either Ets2 binding site reduces the hTERT promoter transcriptional activity. Moreover, Ets2 forms a complex with c-Myc as demonstrated by co-immunoprecipitation and glutathione S-transferase pulldown assays. Immunological depletion of Ets2, or mutation of the EtsA DNA motif, disables c-Myc binding to the E-box, whereas removal of c-Myc or mutation of the E-box also compromises Ets2 binding to EtsA. Thus, hTERT gene expression is maintained by a mechanism involving Ets2 interactions with the c-Myc transcription factor and the hTERT gene promoter, a protein-DNA complex critical for hTERT gene expression and breast cancer cell proliferation.

Chimeric aptamers in cancer cell-targeted drug delivery

Aptamers are single-stranded structured oligonucleotides (DNA or RNA) that can bind to a wide range of targets ("apatopes") with high affinity and specificity. These nucleic acid ligands, generated from pools of random-sequence by an in vitro selection process referred to as systematic evolution of ligands by exponential enrichment (SELEX), have now been identified as excellent tools for chemical biology, therapeutic delivery, diagnosis, research, and monitoring therapy in real-time imaging. Today, aptamers represent an interesting class of modern pharmaceuticals which with their low immunogenic potential mimic extend many of the properties of monoclonal antibodies in diagnostics, research, and therapeutics. More recently, chimeric aptamer approach employing many different possible types of chimerization strategies has generated more stable and efficient chimeric aptamers with aptameraptamer, aptamernonaptamer biomacromolecules (siRNAs, proteins) and aptamernanoparticle chimeras. These chimeric aptamers when conjugated with various biomacromolecules like locked nucleic acid (LNA) to potentiate their stability, biodistribution, and targeting efficiency, have facilitated the accurate targeting in preclinical trials. We developed LNA-aptamer (anti-nucleolin and EpCAM) complexes which were loaded in iron-saturated bovine lactofeerin (Fe-blf)-coated dopamine modified surface of superparamagnetic iron oxide (Fe3O4) nanoparticles (SPIONs). This complex was used to deliver the specific aptamers in tumor cells in a co-culture model of normal and cancer cells. This review focuses on the chimeric aptamers, currently in development that are likely to find future practical applications in concert with other therapeutic molecules and modalities.

EpCAM aptamer mediated cancer cell specific delivery of EpCAM siRNA using polymeric nanocomplex

BACKGROUND: Epithelial cell adhesion molecule (EpCAM) is overexpressed in solid tumors and regarded as a putative cancer stem cell marker. Here, we report that employing EpCAM aptamer (EpApt) and EpCAM siRNA (SiEp) dual approach, for the targeted delivery of siRNA to EpCAM positive cancer cells, efficiently inhibits cancer cell proliferation. RESULTS: Targeted delivery of siRNA using polyethyleneimine is one of the efficient methods for gene delivery, and thus, we developed a novel aptamer-PEI-siRNA nanocomplex for EpCAM targeting. PEI nanocomplex synthesized with EpCAM aptamer (EpApt) and EpCAM siRNA (SiEp) showed 198 nm diameter sized particles by dynamic light scattering, spherical shaped particles, of 151 ± 11 nm size by TEM. The surface charge of the nanoparticles was -30.0 mV using zeta potential measurements. Gel retardation assay confirmed the PEI-EpApt-SiEp nanoparticles formation. The difference in size observed by DLS and TEM could be due to coating of aptamer and siRNA on PEI nanocore. Flow cytometry analysis revealed that PEI-EpApt-SiEp has superior binding to cancer cells compared to EpApt or scramble aptamer (ScrApt) or PEI-ScrApt-SiEp. PEI-EpApt-SiEp downregulated EpCAM and inhibited selectively the cell proliferation of MCF-7 and WERI-Rb1 cells. CONCLUSIONS: The PEI nanocomplex fabricated with EpApt and siEp was able to target EpCAM tumor cells, deliver the siRNA and silence the target gene. This nanocomplex exhibited decreased cell proliferation than the scrambled aptamer loaded nanocomplex in the EpCAM expressing cancer cells and may have potential for EpCAM targeting in vivo.

Synergistic antiproliferative action of the Flavonols Quercetin and Kaempferol in cultured human cancer cell lines

The consumption of vegetables containing the flavonols quercetin and kaempferol reduces the risk of cancer. We utilized human gut (HuTu-80 and Caco-2) and breast cancer cells (PMC42) to show the synergistic effect of quercetin and kaempferol in reducing cell proliferation. A trend in reduction of total cell counts was seen following a single exposure, a 4-day exposure or a 14-day exposure to quercetin and kaempferol. Combined treatments with quercetin and kaempferol were more effective than the additive effects of each flavonol. The reduction in cell proliferation was associated with decreased expression of nuclear proliferation antigen Ki67 and decreased total protein levels in treated cells relative to controls. In conclusion, the synergistic antiproliferative effect of quercetin and kaempferol demonstrated in cultured human cells has broad implications for understanding the influence of dietary nutrients in vivo, where anticancer effects may be a result of nutrients which act in concert.

Multicellular spheroids in ovarian cancer metastases: Biology and pathology

Epithelial ovarian cancer (EOC) has a relatively high mortality rate (~55%). One of the presiding causes is that the current chemotherapeutic regimes are unable to achieve sustained remission, despite frequently producing a positive response at first treatment. One of the reasons that EOC is difficult to treat is that the mechanism of dissemination is unusual. EOC dissemination characteristically involves local invasion of pelvic and abdominal organs. Unlike many epithelial cancers, initial dissemination rarely requires the vasculature, although the vasculature is often implicated in the advanced stages of disease. Recently, it has become apparent that aggregates of malignant cells (spheroids) contained within malignant ascites represent a significant impediment to efficacious treatment of late stage EOC. In vivo, spheroids are present in the malignant ascites of EOC patients, while in vitro cultured spheroids are capable of tumorgenesis in vivo and display a reduced response to chemotherapeutic drugs when compared to monolayers. A major problem associated with the current generation of chemotherapy agents is that they do not address the anchorage and vascular-independent growth conditions associated with a 3-dimensional structure that has formed and/or grown in suspension. Thus, spheroid formation may represent a key component of platinum/taxanesensitive recurrence. If this is correct, a better understanding of spheroid biology may contribute to the identification of new treatment opportunities for the sustained treatment of metastatic EOC. This review article outlines the key biological features of spheroids, specifically discussing their role in EOC dissemination and chemo-response as well as providing insights into spheroid functionality.

Cancer-cell microsurgery using nonlinear optical endomicroscopy

Near-infrared laser-based microsurgery is promising for noninvasive cancer treatment. To make it a safe technique, a therapeutic process should be controllable and energy efficient, which requires the cancer cells to be identifiable and observable. In this work, for the first time we use a miniaturized nonlinear optical endomicroscope to achieve microtreatment of cancer cells labeled with gold nanorods. Due to the high two-photon-excited photoluminescence of gold nanorods, HeLa cells inside a tissue phantom up to 250 μm deep can be imaged by the nonlinear optical endomicroscope. This facilitates microsurgery of selected cancer cells by inducing instant damage through the necrosis process, or by stopping cell proliferation through the apoptosis process. The results indicate that a combination of nonlinear endomicroscopy with gold nanoparticles is potentially viable for minimally invasive cancer treatment.

Selection of DNA aptamers against epithelial cell adhesion molecule for cancer cell imaging and circulating tumor cell capture

Epithelial cell adhesion molecule (EpCAM) is overexpressed in most solid cancers and is an ideal antigen for clinical applications in cancer diagnosis, prognosis, imaging, and therapy. Currently, most of the EpCAM-based diagnostic, prognostic, and therapeutic strategies rely on the anti-EpCAM antibody. However, the use of EpCAM antibody is restricted due to its large size and instability. In this study, we have successfully identified DNA aptamers that selectively bind human recombinant EpCAM protein. The aptamers can specifically recognize a number of live human cancer cells derived from breast, colorectal, and gastric cancers that express EpCAM but not bind to EpCAM-negative cells. Among the aptamer sequences identified, a hairpin-structured sequence SYL3 was optimized in length, resulting in aptamer sequence SYL3C. The Kd values of the SYL3C aptamer against breast cancer cell line MDA-MB-231 and gastric cancer cell line Kato III were found to be 38±9 and 67±8 nM, respectively, which are better than that of the full-length SYL3 aptamer. Flow cytometry analysis results indicated that the SYL3C aptamer was able to recognize target cancer cells from mixed cells in cell media. When used to capture cancer cells, up to 63% cancer cell capture efficiency was achieved with about 80% purity. With the advantages of small size, easy synthesis, good stability, high binding affinity, and selectivity, the DNA aptamers reported here against cancer biomarker EpCAM will facilitate the development of novel targeted cancer therapy, cancer cell imaging, and circulating tumor cell detection.

Targeting cancer cell death using aptamer tagged nano-formulated survivin antagonists

 My findings established survivin antagonist SR9 as an efficient anti-cancer therapeutic and highly promising cancer cell and cancer stem cell targeted locked nucleic acid conjugated nanocarriers as a ray of hope for therapy against colon cancer.