Figure 1.
Englerin A selectively reduces cell viability in renal cancer cells.
(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).
Figure 2.
Englerin A induces cell death morphologically distinct from staurosporine induced apoptosis.
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.
Figure 3.
Englerin A does not lead to up-regulation of extracellular phosphatidyl serine.
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.
Figure 4.
Englerin A does not induce cleavage of caspase 3, PARP, caspase 1 or the autophagic markers LC-3 and Beclin-1.
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.
Figure 5.
Englerin A induces production of reactive oxygen species and increased concentration of intracellular Ca2+.
(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).
Figure 6.
Englerin A sensitivity does not correlate with any known mutations in kidney cancer cell lines.
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. [11] The known mutation status in the cell lines as characterized by Ikediobi and colleagues [44] and as obtained from the Wellcome Trust Sanger Institute Cancer Genome Project web site (http://www.sanger.ac.uk/genetics/CGP) are shown in red (mutated) or green (wild-type).