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Renal cell carcinoma (RCC) is among the top ten most common forms of cancer and is the most common malignancy of the kidney. Clear cell renal carcinoma (cc-RCC), the most common type of RCC, is one of the most refractory cancers with an incidence that is on the rise. Screening of plant extracts in search of new anti-cancer agents resulted in the discovery of englerin A, a guaiane sesquiterpene with potent cytotoxicity against renal cancer cells and a small subset of other cancer cells. Though a few cellular targets have been identified for englerin A, it is still not clear what mechanisms account for the cytotoxicity of englerin A in RCC, which occurs at concentrations well below those used to engage the targets previously identified. Unlike any prior study, the current study used a systems biology approach to explore the mechanism(s) of action of englerin A. Metabolomics analyses indicated that englerin A profoundly altered lipid metabolism by 24 h in cc-RCC cell lines and generated significant levels of ceramides that were highly toxic to these cells. Microarray analyses determined that englerin A induced ER stress signaling and an acute inflammatory response, which was confirmed by quantitative PCR and Western Blot analyses. Additionally, fluorescence confocal microscopy revealed that englerin A at 25 nM disrupted the morphology of the ER confirming the deleterious effect of englerin A on the ER. Collectively, our findings suggest that cc-RCC is highly sensitive to disruptions in lipid metabolism and ER stress and that these vulnerabilities can be targeted for the treatment of cc-RCC and possibly other lipid storing cancers. Furthermore, our results suggest that ceramides may be a mediator of some of the actions of englerin A. Lastly, the acute inflammatory response induced by englerin A may mediate anti-tumor immunity.

ESI (electrospray ionization) MS and tandem mass spectrometry (MS/MS) were used for the analysis of single nucleotide polymorphisms (SNPs) and more complex genetic variations. Double-stranded (ds) PCR products were studied. PCR products of the proline [5'-(mathematical expression not reproducible in ASCII) (G(sub 17))- (mathematical expression not reproducible in ASCII) (C(sub 38)) (mathematical expression not reproducible in ASCII)-3'] and arginine variants [(5'-(mathematical expression not reproducible in ASCII)(G(sub 17))- (mathematical expression not reproducible in ASCII) (G(sub 38)) (mathematical expression not reproducible in ASCII)-3'] of the p53 gene are distinguished by an SNP (cytosine or guanine) and were discriminated using both quadrupole and quadrupole ion trap MS analysis. A 69 bp arginine mutant PCR product [5'-(mathematical expression not reproducible in ASCII)(C(sub 17))- (mathematical expression not reproducible in ASCII)(G(sub 38)) (mathematical expression not reproducible in ASCII)-3'] with a negating switch has the same mass as the proline variant but was readily distinguishable on ion trap MS/MS analysis; fragments containing the mutation site, but not the polymorphism, were identified. The 69 bp PCR products were restriction-enzyme-digested, to create 43 bp fragments. ESI quadrupole ion trap MS/MS analysis of the 43 bp product-ion spectra readily demonstrated both polymorphism and negating switch sites. MS and MS/MS are powerful and complemenmor techniques for analysis of DNA. MS can readily distinguish SNPs but MS/MS is required to differentiate isomeric PCR products (same nucleotide composition but different sequence).

In response to body demands, retinol (ROL) is secreted from the liver into the circulation bound to serum retinol-binding protein (RBP). The mechanism by which ROL is transferred from RBP to target cells remains controversial. To study ROL delivery, we have used a model system of cultured human foreskin keratinocytes (HKc) to compare the uptake, metabolism and biologic effects of ROL added either directly to the medium or bound to RBP. (3H)ROL added directly to the medium was rapidly taken up by HKc, and maximal accumulation of (3H)ROL occurred by 3 h. In contrast, (3H)ROL delivered bound to RBP was taken up very slowly but at a linear rate for at least 72 h. Several experimental approaches indicated that ROL uptake from RBP was not mediated by a cell surface receptor for RBP. The rate and extent of (3H)ROL metabolism to retinyl esters was the same whether the ROL was added directly to the medium or bound to RBP. In addition, several biologic responses by HKc to ROL showed the same dose response curves whether the ROL was added directly to the medium or bound to RBP. Overall, the results support a model of ROL uptake from RBP in which the vitamin is first slowly released from RBP into the aqueous phase and then becomes cell associated. In this manner, the cells are provided with a slow but constant supply of ROL and are protected from the rapid and potentially toxic accumulation of ROL that occurs in the absence of RBP.