Englerin A Selectively Induces Necrosis in Human Renal Cancer Cells

<div><p>The number of renal cancers has increased over the last ten years and patient survival in advanced stages remains very poor. Therefore, new therapeutic approaches for renal cancer are essential. Englerin A is a natural product with a very potent and selective cytotoxicity against renal cancer cells. This makes it a promising drug candidate that may improve current treatment standards for patients with renal cancers in all stages. However, little is known about englerin A's mode of action in targeting specifically renal cancer cells. Our study is the first to investigate the biological mechanism of englerin A action in detail. We report that englerin A is specific for renal tumor cells and does not affect normal kidney cells. We find that englerin A treatment induces necrotic cell death in renal cancer cells but not in normal kidney cells. We further show that autophagic and pyroptotic proteins are unaffected by the compound and that necrotic signaling in these cells coincided with production of reactive oxygen species and calcium influx into the cytoplasm. As the first study to analyze the biological effects of englerin A, our work provides an important basis for the evaluation and validation of the compound's use as an anti-tumor drug. It also provides a context in which to identify the specific target or targets of englerin A in renal cancer cells.</p> </div

Organometallic Titanocene–Gold Compounds as Potential Chemotherapeutics in Renal Cancer. Study of their Protein Kinase Inhibitory Properties

Early–late transition metal TiAu₂ compounds [(η-C₅H₅)₂Ti­{OC­(O)­CH₂PPh₂AuCl}₂] (<b>3</b>) and new [(η-C₅H₅)₂Ti­{OC­(O)-4-C₆H₄­PPh₂AuCl}₂] (<b>5</b>) were evaluated as potential anticancer agents <i>in vitro</i> against renal and prostate cancer cell lines. The compounds were significantly more effective than monometallic titanocene dichloride and gold­(I) [{HOC­(O)­RPPh₂}­AuCl] (R = −CH₂– <b>6</b>, −4-C₆H₄– <b>7</b>) derivatives in renal cancer cell lines, indicating a synergistic effect of the resulting heterometallic species. The activity on renal cancer cell lines (for <b>5</b> in the nanomolar range) was considerably higher than that of cisplatin and highly active titanocene Y. Initial mechanistic studies in Caki-1 cells <i>in vitro</i> coupled with studies of their inhibitory properties on a panel of 35 kinases of oncological interest indicate that these compounds inhibit protein kinases of the AKT and MAPKAPK families with a higher selectivity toward MAPKAPK3 (IC₅₀ <b>3</b> = 91 nM, IC₅₀ <b>5</b> = 117 nM). The selectivity of the compounds <i>in vitro</i> against renal cancer cell lines when compared to a nontumorigenic human embryonic kidney cell line (HEK-293T) and the favorable preliminary toxicity profile on C57black6 mice indicate that these compounds (especially <b>5</b>) are excellent candidates for further development as potential renal cancer chemotherapeutics

Englerin A induces production of reactive oxygen species and increased concentration of intracellular Ca²⁺.

<p>(A) Cells were treated with either 1 μM englerin A or carrier DMSO for 60 min. The relative change in reactive oxygen (ROS) or reactive nitrogen species (RNS) compared to cells treated with the carrier DMSO was measured using the Total ROS detection kit. Histograms show fluorescence intensities in a representative experiment (left panel). Quantified relative changes in ROS/RNS shown (right panel) are means ± SEM (n = 5), statistically significant differences are marked with asterisks (* p<0.05). (B) Cells were treated with either 1 μM englerin A or carrier DMSO for 60 min, or 10 μM ionomycin for 50 min. Fluo-3 binding to Ca²⁺ ions was measured through an increased fluorescence emission of the dye at 520 nm upon excitation at 485 nm. Histograms show fluorescence intensities in a representative experiment (left panel). Quantified relative changes in intracellular calcium ions shown (right panel) are means ± SEM (n = 3), statistically significant differences are marked with asterisks (* p<0.05, *** p<0.001).</p

Englerin A does not lead to up-regulation of extracellular phosphatidyl serine.

<p>Cells were treated with either 1 μM englerin A or carrier DMSO for 60 min, or 5 μM staurosporine for 3 h. After incubation, cells were trypsinized and stained for extracellular phosphatidyl serine expression using FITC-tagged Annexin V and propidium iodide (PI) as co-stain to test cell membrane integrity. Shown is a result representative of three independent experimental repeats. Quantifications and statistics of all data are depicted as bar graphs and show the distribution of cells testing positive for Annexin V binding (early apoptotic stages) or Annexin V binding and propidium iodide uptake (late apoptotic stages/necrotic death). Values shown are mean ± SEM (n = 3), statistically significant differences are marked with asterisks (*** p<0.001), n.s. = not significant.</p

Englerin A induces cell death morphologically distinct from staurosporine induced apoptosis.

<p>Micrographs show the morphology of cells treated with englerin A or staurosporine, a known inducer of apoptosis. Cells were treated with either 1 μM englerin A or carrier DMSO for 60 min, or 1 μM staurosporine for 5 h. Pictures were taken using a Zeiss Axiovert200 M microscope with a 40× phase objective. For every treatment, 5–10 random fields of vision were acquired. The experiment was repeated three times, micrographs shown are representative of the average cell morphology upon treatment. Scale bars represent 20 μm.</p

Englerin A selectively reduces cell viability in renal cancer cells.

<p>(A) Chemical Structure of englerin A. (B) Glioblastoma (SF-295), normal immortalized kidney cells (HEK-293), renal proximal tubule cells (RPTC) and renal cancer cells (UO-31, A-498) were incubated with the indicated concentration of englerin A for 48 h. Cell viability was analyzed using an XTT Cell Proliferation Assay. Results are shown in % viability compared to a cell sample treated with the carrier DMSO. Values shown represent the mean ± SEM of all experiments (n≥6). IC50 values were calculated with Prism 5 using a non-linear regression fit (log(inhibitor) vs. normalized response – variable slope).</p

Englerin A sensitivity does not correlate with any known mutations in kidney cancer cell lines.

<p>Renal cell carcinoma cell lines from the NCI-60 cell panel and a glioblastoma cell line (SF-295) were arranged by decreasing sensitivity to the englerin A natural product as determined by Akee and colleagues. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048032#pone.0048032-Akee1" target="_blank">[11]</a> The known mutation status in the cell lines as characterized by Ikediobi and colleagues <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048032#pone.0048032-Ikediobi1" target="_blank">[44]</a> and as obtained from the Wellcome Trust Sanger Institute Cancer Genome Project web site (<a href="http://www.sanger.ac.uk/genetics/CGP" target="_blank">http://www.sanger.ac.uk/genetics/CGP</a>) are shown in red (mutated) or green (wild-type).</p

Englerin A does not induce cleavage of caspase 3, PARP, caspase 1 or the autophagic markers LC-3 and Beclin-1.

<p>Cells were treated with either 1 μM englerin A, carrier DMSO or 5 μM staurosporine for the indicated amount of time. (A) After the incubation, cells were lysed and lysates were analyzed by immunoblotting for PARP cleavage or full-length and cleaved caspase 3 (Casp3-fl, Casp3-cl). Equal protein loading was confirmed by probing for GAPDH. Full-length and cleaved bands are indicated. The experiment was repeated three times. (B) Alternatively, after incubation cells were lysed and caspase 3 activity was tested using a caspase 3 activity assay kit. Values shown are means ± SEM (n = 6), statistically significant differences are marked with asterisks (*** p<0.001). (C) Cells were treated with either 1 μM englerin A, carrier DMSO for 60min or 50 μM chloroquine diphosphate (Chloro) for 18 h. After the incubation, cells were lysed and lysates were analyzed by immunoblotting for Beclin-1, LC3-I/II and caspase 1 cleavage (proenzyme p45 and cleaved active subunit p20). Equal protein loading was confirmed by probing for GAPDH. All membranes were analyzed using IRDye secondary antibodies and a Licor Odyssey system. Membranes shown are from representative experiments.</p