Effect of lanatoside C and tumor necrosis factor–related apoptosis-inducing ligand (TRAIL) on glioblastoma multiforme (GBM) cells. (A) Validation of lanatoside C on U87 and Gli36 cell lines, GBM1 primary cells, and primary human fibroblasts (HF19), all expressing Gluc-CFP. Cells were treated with lanatoside C (0.25 µM) and/or TRAIL (50 ng/mL), and Gluc activity was measured after 48 h. (B) Two different primary GBM cells dissociated from different patients and primary human fibroblasts were treated with 0.25 µM of lanatoside C and 50 ng/mL TRAIL. Aliquots of the conditioned medium were assayed for Gluc activity over time. (C) Half-maximal inhibitory concentration (IC₅₀) of lanatoside C in combination with TRAIL. Primary GBM cells were treated with different concentrations of lanatoside C and TRAIL (50 ng/mL). Gluc activity was assayed in conditioned medium at 24, 48, and 72 h. (D) CFP fluorescence images of GBM cells treated with different doses of lanatoside C and/or TRAIL at 24, 48, and 72 h. (E) Effect of lanatoside C alone on GBM cells. Various GBM cells were treated in a 96-well plate with various amounts of lanatoside C. Gluc activity was assayed 48 h later. Data are presented as the percentage of Gluc expression in which the control untreated sample is set at 100%. The average of experimental triplicates ± standard deviation is shown. *P≤ .05, by the Student's t test. **P≤ .001, by the Student's t test.

(A) Lanatoside C upregulates DR5 on GBM cells. U87 cells were treated with 0.25 or 1 µM of lanatoside C. Sixteen hours later, cells were labeled with anti DR4- or DR5-PE–conjugated antibodies followed by FACS analysis. Data are shown as mean fluorescence intensity (MFI) ± standard deviation. (B) U87-Gluc-CFP cells were pretreated with tumor necrosis factor–related apoptosis-inducing ligand (TRAIL)–R2-Fc chimeric protein (300 ng/mL) followed by lanatoside C in the presence or absence of TRAIL. Cell viability was assayed using CellTiter-Glo 24 h later. Data are presented as the percentage of cell viability in treated versus untreated samples and are shown as the mean of biological triplicates ± standard deviation.

Effect of Bcl-2 overexpression on lanatoside C–induced cell death. U87 cells stably expressing a control vector, Bcl-2, or Bcl-xL were treated with different concentrations of lanatoside C (A), lanatoside C and tumor necrosis factor–related apoptosis-inducing ligand (TRAIL) (B) or staurosporine (STS; positive control) (C). Twenty-four hours later, cell viability was assayed using CellTiter-Glo. The upper panel shows western blot analysis for overexpression of Bcl2 or Bcl-xl in these cells.

Lanatoside C-induces mitochondrial damage and ATP depletion. (A) U87 cells were treated with 0.25 µM (left panel) or 1 µM (middle panel) lanatoside C. Four hours later, mitochondrial membrane potential (MMP) was measured after JC-1 staining, followed by flow cytometric analysis. The right panel represents quantitation analysis of (A) showing the percentage of cells with low MMP. (B) Time-course analysis of ATP levels following treatment of U87 cells with PBS or lanatoside C (1 µM) using CellTiter-Glo assay. All experiments were repeated at least 3 times to obtain statistical significance, *P≤ .05, by the Student's t test. **P≤ .001, by the Student's t test.

Effect of lanatoside C and/or tumor necrosis factor–related apoptosis-inducing ligand (TRAIL) on glioblastoma multiforme (GBM) tumors in vivo. U87 glioma cells expressing Gluc were implanted subcutaneously in nude mice. One week later, mice were intraperitoneally injected once per day over 10 days with either DMSO vehicle (control), TRAIL (250 µg/kg body weight), lanatoside C (6 mg/kg body weight), or similar doses of both lanatoside C and TRAIL. (A) In vivo Gluc bioluminescence imaging was performed once per week after intravenous injection of coelenterazine, and photon counts were acquired using a CCD camera. (B) Before and at different time points (twice per week) after treatment, 5 µL of blood was drawn and assayed for Gluc activity. Shown is a representative mouse from each group (A) and the  mean ± standard deviation for Gluc blood assay from each group (B; n = 10).

Lanatoside C induces caspase-independent cell death. (A) U87 cells were treated for 6 h with lanatoside C (0.25 and 1 µM), tumor necrosis factor–related apoptosis-inducing ligand (TRAIL; 50 ng/mL), or a combination of both and visualized using transmission electron microscopy (scale bar, 2 µm; original magnification, ×4000). Arrowheads indicate condensed chromatin; arrows indicate vacuoles present in the cytosol. (B) U87 cells treated for 16 h with lanatoside C (0.25 or 1 µM) in the presence or absence of TRAIL (50 ng/mL) were harvested and analyzed by western blotting using cleaved caspases 3 and 8, BID, PARP, and pro-caspase 9 as well as β-actin specific antibodies. (C and D) U87 subcutaneous tumor xenografts received injections of DMSO, lanatoside C, TRAIL, or a combination of lanatoside C and TRAIL. Tumors were dissociated, and lysates were analyzed using caspase 3/7-Glo assay (C) or for BID expression using western blotting (D). The ratio of BID to β-actin is shown below the blot, with values normalized to the control sample. (E) U87 cells were treated with either lanatoside C and TRAIL or staurosporine (STS; positive control) in the presence or absence of the caspase inhibitor zVAD-FMK (100 µM, with 1 h of pretreatment). Cell viability was assayed 24 h later using CellTiter-Glo.

Lanatoside C induces autophagy. (A) Transmission electron micrograph of an untreated cell (original magnification, ×4000) or treated with 1 µM of lanatoside C (original magnification, ×8000). Arrows depict autophagosomes containing intracellular organelles (scale bar, 2 µm). (B) U87 cells treated for 16 h with lanatoside C (0.25 or 1 µM) were harvested and analyzed by western blotting using LC3 and β-actin antibodies. The ratio of LC3 II to β-actin is shown below the blot, with values normalized to the control sample. (C) U87 cells stably expressing LC3-GFP were treated with PBS, lanatoside C (1 µM), rapamycin (200 nM), or rapamycin (200 nM) in combination with 3MA (2 mM) for 24 h. Representative GFP fluorescent microscopic images of cells counterstained with DAPI (nuclei) are shown. Scale bar, 10 µm.