Inhibition of the MUC1-C oncoprotein is synergistic with cytotoxic agents in the treatment of breast cancer cells

Figures & data

Figure 1. Combining GO-203 with taxol and DOX increases the induction of MCF-7 cell apoptosis. (A) Schema of the MUC1-C subunit with the sequence of the 72-amino acid cytoplasmic domain. GO-203 is a D-amino acid peptide that includes a poly-Arg transduction domain and the CQCRRKN sequence (shaded) derived from the MUC1-C cytoplasmic domain. (B) MCF-7 cells were left untreated (control) and treated with 5.6 μM GO-203 (Table 1), 28 nM taxol (Table 1) or the combination of both agents for 72 h. GO-203 was added every 24 h. (C) MCF-7 cells were left untreated (control) and treated with 5.6 M GO-203, 0.33 μM DOX (Table 1) or the combination of both agents for 72 h. GO-203 was added every 24 h. The cells were fixed and analyzed for cell cycle distribution by flow cytometry. The percentage of sub-G₁ cells is included in the panels.

Table 1. IC₅₀ values of GO-203, taxol and DOX

Cell line	GO-203 (μM)	Taxol (nM)	DOX (μM)
MCF-7	5.6 ± 0.5	28 ± 12	0.33 ± 0.06
ZR-75–1	2.9 ± 0.4	85 ± 36	0.53 ± 0.04

Determination of IC₅₀ values. The indicated cells were seeded on 96-well plates at a density of 4,000 cells/well. After 24 h, the cells were exposed to varying concentrations of GO-203 (added every 24 h), taxol or DOX. After incubation for 72 h, viability was determined using the alamar blue viability assay. The percentage of viable cells was determined as compared with that obtained with control untreated cells. The IC₅₀ (concentration that inhibits viability by 50%) was determined by nonlinear regression of the dose-response data using Prism 5.0 for MAC OSX. Each experiment was performed in triplicate and repeated three times. The IC₅₀ value is expressed as the mean ± SD.

Figure 2. Activation of effector caspase-7 in MCF-7 cells treated with GO-203, taxol and DOX. (A) MCF-7 cells were treated with 5.6 μM GO-203, 28 nM taxol or the combination of both agents for 48 h. GO-203 was added every 24 h. B. MCF-7 cells were treated with 5.6 μM GO-203, 0.33 μM DOX, or the combination of both agents for 48 h. GO-203 was added every 24 h. Cytosolic lysates were immunoblotted with the indicated antibodies (left). Total cell lysates were immunoblotted with anti-PARP and anti-actin (right). FL, full-length; CF, cleaved fragment.

Figure 3. GO-203 increases the apoptotic response of ZR-75-1 cells to taxol and DOX. (A) ZR-75-1 cells were left untreated (control) and treated with 2.9 μM GO-203 (Table 1), 85 nM taxol (Table 1) or the combination of both agents for 72 h. GO-203 was added every 24 h. (B) ZR-75-1 cells were left untreated (control) and treated with the combination of 2.9 μM GO-203 and 85 nM taxol in the absence and presence of 4 μM Z-VAD-FMK for 48 h. GO-203 was added every 24 h. (C) ZR-75-1 cells were left untreated (control) and treated with 2.9 μM GO-203, 0.53 μM DOX (Table 1) or the combination of both agents for 72 h. GO-203 was added every 24 h. (D) ZR-75-1 cells were left untreated (control) and treated with the combination of 2.9 M GO-203 and 0.53 μM DOX in the absence and presence of 4 μM Z-VAD-FMK for 48 h. GO-203 was added every 24 h. The cells were fixed and analyzed for cell cycle distribution by flow cytometry. The percentage of sub-G₁ cells is included in the panels.

Figure 4. Activation of caspase-3 and caspase-7 in ZR-75-1 cells treated with GO-203 in combination with taxol and DOX. (A) ZR-75-1 cells were treated with 2.9 μM GO-203, 85 nM taxol or the combination of both agents for 48 h. GO-203 was added every 24 h. (B) ZR-75-1 cells were treated with 2.9 μM GO-203, 0.53 μM DOX or the combination of both agents for 48 h. GO-203 was added every 24 h. Cytosolic lysates were immunoblotted with the indicated antibodies (left). Total-cell lysates were immunoblotted with anti-PARP and anti-actin (right). FL, full-length; CF, cleaved fragment.

Figure 5. Combining GO-203 with taxol or DOX induces late apoptosis/necrosis. (A) ZR-75-1 cells were treated with 2.9 μM GO-203, 85 nM taxol or the combination of both agents for 48 h. GO-203 was added every 24 h. (B) ZR-75-1 cells were treated with 2.9 μM GO-203, 0.53 μM DOX or the combination of both agents for 48 h. GO-203 was added every 24 h. The cells were then incubated with PI/annexin V and analyzed by flow cytometry. The percentage of necrotic (upper left quadrant), apoptotic (lower right quadrant) and late apoptotic/necrotic (upper right quadrant) cells is indicated in the panels.

Figure 6. Synergistic interaction of GO-203 in combination with taxol and DOX in the treatment of MCF-7 cells. (A) MCF-7 cells were exposed to fixed IC₅₀ ratios GO-203 alone, taxol alone and the GO-203/taxol combination. The dose-effect curves for GO-203 alone (+), taxol alone (8) and GO-203/taxol (X) are shown in the left panel. The multiple effect-level isobologram analysis is shown in the right panel for the ED₉₀ (solid circles), ED₇₅ (solid squares) and ED₅₀ (solid triangles). The CI values are listed in Table 2. (B) MCF-7 cells were treated with fixed IC₅₀ ratios of GO-203 alone, DOX alone and the GO-203/DOX combination. The dose-effect curves for GO-203 alone (+), DOX alone (8) and GO-203/DOX (X) are shown in the left panel. The multiple effect-level isobologram analysis is shown in the right panel for the ED₉₀ (solid circles), ED₇₅ (solid squares) and ED₅₀ (solid triangles). The CI values are listed in Table 2.

Table 2. Combination indices

CI value for GO-203 and Taxol
Cell line	ED50	ED75	ED90
MCF-7	0.836	0.711	0.676
ZR-75-1	0.760	0.731	0.802
CI value for GO-203 and DOX
Cell line	ED50	ED75	ED90
MCF-7	0.630	0.777	1.097
ZR-75-1	0.589	0.448	0.341

Determination of combination indices. The indicated cells were exposed to 1:1 ratios of the respective IC₅₀ values of GO-203 (added every 24 h) in combination with taxol or DOX as described in the Materials and Methods. Cell viability was determined after treatment for 72 h using the alamar blue assay. Combination indices (CIs) were determined at the effective dose (ED) 50, 75 and 90 for each of the drugs using CalcuSyn software.

Figure 7. GO-203 is synergistic in combination with taxol and DOX in the ZR-75-1 cell model. (A) ZR-75-1 cells were exposed to fixed IC₅₀ ratios of GO-203 alone, taxol alone and the GO-203/taxol combination. The dose-effect curves for GO-203 alone (+), taxol alone (8) and GO-203/taxol (X) are shown in the left panel. The multiple effect-level isobologram analysis is shown in the right panel for the ED₉₀ (solid circles), ED₇₅ (solid squares) and ED₅₀ (solid triangles). The CI values are listed in Table 2. (B) ZR-75-1 cells were treated with fixed IC₅₀ ratios of GO-203 alone, DOX alone and the GO-203/DOX combination. The dose-effect curves for GO-203 alone (+), DOX alone (8) and GO-203/DOX (X) are shown in the left panel. The multiple effect-level isobologram analysis is shown in the right panel for the ED₉₀ (solid circles), ED₇₅ (solid squares) and ED₅₀ (solid triangles). The CI values are listed in Table 2.