RAGE overexpression confers a metastatic phenotype to the WM115 human primary melanoma cell line.

The formation of melanoma metastases from primary tumor cells is a complex phenomenon that involves the regulation of multiple genes. We have previously shown that the receptor for advanced glycation end products (RAGE) was up-regulated in late metastatic stages of melanoma patient samples and we hypothesized that up-regulation of RAGE in cells forming a primary melanoma tumor could contribute to the metastatic switch of these cells. To test our hypothesis, we overexpressed RAGE in the WM115 human melanoma cell line that was established from a primary melanoma tumor of a patient. We show here that overexpression of RAGE in these cells is associated with mesenchymal-like morphologies of the cells. These cells demonstrate higher migration abilities and reduced proliferation properties, suggesting that the cells have switched to a metastatic phenotype. At the molecular level, we show that RAGE overexpression is associated with the up-regulation of the RAGE ligand S100B and the down-regulation of p53, ERK1/2, cyclin E and NF-kB. Our study supports a role of RAGE in the metastatic switch of melanoma cells.

pH-triggered echogenicity and contents release from liposomes.

Liposomes are representative lipid nanoparticles widely used for delivering anticancer drugs, DNA fragments, or siRNA to cancer cells. Upon targeting, various internal and external triggers have been used to increase the rate for contents release from the liposomes. Among the internal triggers, decreased pH within the cellular lysosomes has been successfully used to enhance the rate for releasing contents. However, imparting pH sensitivity to liposomes requires the synthesis of specialized lipids with structures that are substantially modified at a reduced pH. Herein, we report an alternative strategy to render liposomes pH sensitive by encapsulating a precursor which generates gas bubbles in situ in response to acidic pH. The disturbance created by the escaping gas bubbles leads to the rapid release of the encapsulated contents from the liposomes. Atomic force microscopic studies indicate that the liposomal structure is destroyed at a reduced pH. The gas bubbles also render the liposomes echogenic, allowing ultrasound imaging. To demonstrate the applicability of this strategy, we have successfully targeted doxorubicin-encapsulated liposomes to the pancreatic ductal carcinoma cells that overexpress the folate receptor on the surface. In response to the decreased pH in the lysosomes, the encapsulated anticancer drug is efficiently released. Contents released from these liposomes are further enhanced by the application of continuous wave ultrasound (1 MHz), resulting in substantially reduced viability for the pancreatic cancer cells (14%).

Performance Characterization and Validation of Saliva as an Alternative Specimen Source for Detecting Hereditary Breast Cancer Mutations by Next Generation Sequencing.

Identification of pathogenic germline mutations by next generation sequencing is a widely accepted tool for predicting the risk of hereditary cancer development. Blood is the most common source of DNA for such tests. However, blood as a sample type has many drawbacks, including the invasive collection method, poor sample stability, and a relatively high cost of collection. Therefore, in the current study we have assessed the suitability of saliva as an alternative source of genomic DNA for the identification of germline mutations in the BRCA1/2 genes by next generation sequencing (NGS). Our results show that all of the samples yielded DNA concentrations sufficient for library preparation. The concentrations of the final libraries, which were generated by PCR using target specific primers, fall into the expected range with no notable difference between libraries generated from DNA derived from saliva or blood. Quality parameters indicate that sequencing performance is comparable across sample source. An average of (98 ± 0.02)% variant calling concordance was obtained between the two specimen sources. Our data recommends saliva as a potential alternative for detecting germline mutation by next generation sequencing.

The receptor for advanced glycation end products influences the expression of its S100 protein ligands in melanoma tumors.

Recent studies have suggested that the receptor for advanced glycation end products (RAGE) participates in melanoma progression by promoting tumor growth. However, the mechanisms of RAGE activation in melanoma tumors are not clearly understood. To get deeper insights into these mechanisms, we transfected a melanoma cell line, which was established from a human melanoma primary tumor, with RAGE, and studied the effect of RAGE overexpression on cell proliferation and migration in vitro. We observed that overexpression of RAGE in these cells not only resulted in significantly increased migration rates compared to control cells, but also in decreased proliferation rates (Meghnani et al., 2014). In the present study, we compared the growth of xenograft tumors established from RAGE overexpressing WM115 cells, to that of control cells. We observed that when implanted in mice, RAGE overexpressing cells generated tumors faster than control cells. Analysis of protein tumor extracts showed increased levels of the RAGE ligands S100B, S100A2, S100A4, S100A6 and S100A10 in RAGE overexpressing tumors compared to control tumors. We show that the tumor growth was significantly reduced when the mice were treated with anti-RAGE antibodies, suggesting that RAGE, and probably several S100 proteins, were involved in tumor growth. We further demonstrate that the anti-RAGE antibody treatment significantly enhanced the efficacy of the alkylating drug dacarbazine in reducing the growth rate of RAGE overexpressing tumors.

Multifunctional polymersomes for cytosolic delivery of gemcitabine and doxorubicin to cancer cells.

Although liposomes are widely used as carriers of drugs and imaging agents, they suffer from a lack of stability and the slow release of the encapsulated contents at the targeted site. Polymersomes (vesicles of amphiphilic polymers) are considerably more stable compared to liposomes; however, they also demonstrate a slow release for the encapsulated contents, limiting their efficacy as a drug-delivery tool. As a solution, we prepared and characterized echogenic polymersomes, which are programmed to release the encapsulated drugs rapidly when incubated with cytosolic concentrations of glutathione. These vesicles encapsulated air bubbles inside and efficiently reflected diagnostic-frequency ultrasound. Folate-targeted polymersomes showed an enhanced uptake by breast and pancreatic-cancer cells in a monolayer as well as in three-dimensional spheroid cultures. Polymersomes encapsulated with the anticancer drugs gemcitabine and doxorubicin showed significant cytotoxicity to these cells. With further improvements, these vesicles hold the promise to serve as multifunctional nanocarriers, offering a triggered release as well as diagnostic ultrasound imaging.