Dependency on Bax and Bcl-2.

<p>(A, B, F) Mitochondrial membrane potential (Δψm) was measured in A-375, A-375-TS, mock and IK1-transfected HEK-293, A375-Mock and A375-Bcl-2 at 24 h and 48 h of treatment with TRAM-34 (40 µM) +/− TRAIL. Treated cells (open graphs) were compared with DMSO-treated controls (grey). Each three independent experiments showed comparable results. (C) Expression of Bcl-2 proteins was determined by Western blotting in A-375 and A-375-TS in response to TRAM-34 (40 µM) +/− TRAIL. Two independent experiments showed comparable results. (D, E) Apoptosis induced by TRAM/TRAIL was investigated in (D) HCT-116 parental cells (Bax⁺, Bak⁺) and in subclones with knockdown for Bax and/or Bak, as well as in (E) A-375 cells stably transfected with Bcl-2 (A375-Bcl-2) and mock transfected controls. Expression of Bcl-2 proteins was controlled by Western blotting (insets). Statistical significance is indicated (*; p<0.005) for comparison of parental cells and mock controls.</p

Death receptor expression in response to TRAM-34.

<p>(A, B) Expression of DR5 is shown in five melanoma cell lines in response to TRAM-34 (40 µM, +), and (C, D) expression of DR4 is shown in A-375 and A-375-TS, as compared to DMSO-treated controls (−). Total protein expression was determined by Western blotting, and surface expression was determined by flow cytometry. (E) Expression of DcR1 and DcR2 is shown by flow cytometry in five melanoma cell lines in response to TRAM-34. HeLa and SW480 cells served as positive controls for DcR1 and DcR2, respectively. IgG1-stained cells served as negative controls. Three (A, C) or two (E) independent experiments, with each time triplicates revealed highly similar results. (F) Apoptosis in response to TRAM/TRAIL was monitored in A-375 and Mel-HO after blocking DR4 and/or DR5 by selective antagonistic antibodies (mean values and SDs of two independent experiments, each one with triplicates). Statistical significance is indicated (*; p<0.005), when comparing combined treatment with TRAIL alone.</p

Dependency on XIAP, Bid and SMAC.

<p>(A, C, D) The sensitivity of A-375 and A-375-TS cells for TRAM/TRAIL-induced apoptosis is shown (A) after siRNA-mediated Bid knockdown, (C) plasmid-mediated XIAP overexpression and (D) siRNA-mediated SMAC knockdown. The respective mock controls are shown for comparison. Apoptosis (% of sub-G1 cells) and cytotoxicity (LDH release) is shown at 24 h of treatment. Mean values and SDs of three (XIAP, SMAC) or two independent experiments (Bid) are shown; each experiment consisted of triplicates. Statistical significance is indicated (*; p<0.005), when comparing to the respective mock controls. Overexpression or downregulation is shown in insets. (B) Expression of cIAPs (survivin, XIAP and cIAP-2) as well as of SMAC was analyzed by Western blotting in total protein extracts of melanoma cell lines.</p

Sensitization of melanoma cells for TRAIL-induced apoptosis.

<p><b>(</b>A) Apoptosis (percentage of sub-G1 cells) was determined by cell cycle analyses in nine melanoma cell lines treated with increasing concentrations of TRAM-34+/− TRAIL. (B) Examples are shown of A-375 and A-375-TS treated with the combination of 40 µM TRAM-34+ TRAIL (open graph) as compared to DMSO-treated controls (filled graph). (C, D) For A-375 and A-375-TS, cell proliferation was determined by WST-1 (C) and cytotoxicity by LDH-release (D). (E) Apoptosis was monitored after treatment with two concentrations of charybdotoxin (CTX) +/− TRAIL. (F) Induction of apoptosis by TRAM-34+/− TRAIL was determined in mock- and IK1-transfected HEK-293 cells. Protein expression of IK1 at 24 h after transfection is shown in the inset, as determined by Western blotting. (A, C, D, E, F) Means and SDs are shown of three independent experiments, each one consisting of triplicates. Statistical significance (*; p<0.005) is indicated, when comparing TRAM/TRAIL-treated cells with TRAIL-treated cells.</p

Synergism studies for TRAM-34 and TRAIL.

<p>(A) Induction of apoptosis (sub-G1 cells) is shown in four melanoma cell lines in response to TRAM-34 (20–80 µM) and TRAIL (10–40 ng/ml), given alone or in combination. Calculated CI values are given in parenthesis above the bars of the combinations. For dose effect analyses, relative concentrations of TRAM-34 were plotted against relative concentrations of TRAIL shown in normalized isobolograms (insets). The hypotenuse indicates the line of additive effects, whereas values below are synergistic. Three independent experiments revealed largely similar results.</p

Antiproliferative effects by IK1 inhibition.

<p>(A) Protein expression of IK1 is shown in six melanoma cell lines. Negative control: mock-transfected HEK-293; positive control: IK1-transfected HEK-293 cells. Equal protein loading (30 µg per lane) is proven by GAPDH. (B) Expression of IK1 mRNA in melanoma cell lines was determined by real-time PCR. The threshold of negative HEK-293 is indicated. Values were normalized to β-actin. (A, B) Each two independent experiments revealed comparable results. (C) Voltage-dependent potassium currents were recorded in TRAM-34-treated A-375, as compared to non-treated controls. The voltage range, used for subsequent time course, is indicated. (D) Time course of potassium currents in A-375 after addition of TRAM-34 (t = 0) is shown. Short arrowheads indicate the time interval for determination of the current/voltage dependency. (E) Cell cycle analysis of TRAM-34-treated A-375 as compared to DMSO-treated controls. (F) Decreased proliferation in response to increasing concentrations of TRAM-34 is shown for Mel-HO, as determined by WST-1 assay. Means and SDs are shown of three independent experiments, each one consisting of triplicates. Statistical significance is indicated (*; p<0.005), when comparing TRAM-34-treated cells with DMSO-treated controls. (G, H) Real-time growth curves of TRAM-34-treated A-375 and Mel-HO are compared to DMSO-treated controls. Cell indices were normalized at t = 0.</p

Caspase activation in response to TRAM and TRAIL.

<p>(A) Processing of caspase-8, -9 and -3 in A-375 and A-375-TS in response to TRAM-34 (40 µM) and TRAIL is shown by Western blotting. Two independent experiments revealed comparable results. (B) Apoptosis (% of sub-G1 cells) in A-375 and A-375-TS is shown in response to TRAIL, TRAM-34 and the pancaspase inhibitor Q-VD-OPh (10 µM, 1 h pretreatment). (C) Cell proliferation was determined by WST-1. (B, C) Mean values and SDs are shown of two independent experiments, each one with triplicates. Statistical significance of abrogated apoptosis and recovered cell proliferation by Q-VD-OPh is indicated (*; p<0.005).</p

Mitochondrial release of proapoptotic factors.

<p>(A) Mitochondrial extracts of A-375, treated as indicated with TRAM-34 and TRAIL for 24 h, were analyzed for IK1 by Western blotting. Comparable amounts of mitochondrial extracts were loaded as proven by incubation with prohibitin antibody. A cytosolic extract (Cyto) as well as total protein extracts of mock or IK1-transfected HEK-293 served as controls. (B, C) Mitochondrial (Mito) and cytosolic extracts (Cyto) of A-375 and A-375-TS treated for 4 h with TRAM-34 (40 µM) +/−TRAIL were investigated by Western blotting. Equal loading was proven by the mitochondrial protein VDAC and cytosolic protein GAPDH, respectively. VDAC applied to cytosolic extracts ruled out contaminations with mitochondria.</p