Article Figures & Data
Figures
- Figure 1.
Identification of propachlor as a collaborating compound with Rad001. A, five thousand compounds were added alone or together with 20 nmol/L Rad001 in C4-2 cells, the survival ratio gauged by ATP measurement between cells that are treated with single compound or combination with Rad001 is plotted. B, the chemical structures of Rad001 and propachlor. C, propachlor can collaborate with Rad001 to inhibit PC3 and C4-2 cells. Single compounds or combination with Rad001 were used to treat PC3 or C4-2 cells for 72 hours followed by CellTiter-Glo measurement to measure cell number. The 2-way t test showed P < 0.05 between the 2 groups.
- Figure 2.
The combination of Rad001 and propachlor induces a synergistic reduction of cell number in prostate cancer cells. A, decreasing doses of Rad001 alone, propachlor alone, and their combination were used, and cell numbers measured as in Fig. 1 (x-axis, Rad001 μmol/L; y-axis, propachlor μmol/L; z-axis, 1 − (treatment luminescence unit/control luminescence). B, normalized isobologram analysis showed synergistic interactions in PC3 and C4-2 cells. The analysis was done with CalcuSyn software, which conducts the drug dose–effect calculation with the median effect method described by Chou (7). A large number of combination groups were below the line, indicating synergism. C, growth curves by measuring ATP level shows more reduction of cell number when cells were treated with the drug combination.
- Figure 3.
Combination of Rad001 and propachlor synergistically induces cell death in prostate cancer cells. PC3 cells were treated with DMSO (control), Rad001 (0.70 μmol/L), propachlor (6.15 μmol/L), or their combination, and C4-2 cells were treated with DMSO (control), Rad001 (0.56 μmol/L), propachlor (7.05 μmol/L), or their combination. A, trypan blue (TB) assay. Combination treatment resulted in lower cell viability than either compound alone (2-way t test: P < 0.005). B, increased levels of cleaved PARP1 and caspase-3 and decreased Bcl-2 in PC3 and C4-2 cells treated for 15 hours with the combination of Rad001 and propachlor. Lysates from cells were separated by SDS-PAGE followed by immunoblotting with respective antibodies. C, higher activities of caspase-3/7 in PC3 and C4-2 cells treated for 15 hours with the drug combination. D, flow cytometric analysis of apoptosis with Annexin-V and 7-AAD staining. There was a significant increase in the percentage of apoptotic cell after combination treatment versus single compound (P < 0.05).
- Figure 4.
Rad001 and propachlor combination synergistically induces autophagy in PC3 and C4-2 cells. Autophagy was analyzed after 1 day of treatment. PC3 cells were treated with DMSO (control), Rad001 (0.70 μmol/L), propachlor (6.15 μmol/L), or their combination, and C4-2 cells were treated with DMSO (control), Rad001 (0.56 μmol/L), propachlor (7.05 μmol/L), or their combination. A, upregulation of LC3-2 in PC3 and C4-2 cells treated with combination of the 2 compounds. Immunoblotting was conducted as in Fig. 3. B, increased autophagosomes (arrows) were observed by electron microscopy in cells treated with Rad001/propachlor compared with each compound alone. Quantified data from 30 cells are shown in the bottom. C, autophagosome analysis through GFP-LC3 expression. C4-2 and PC3 cells expressing GFP-LC3 were treated with DMSO, Rad001, propachlor, or their combination for 24 hours. The cells were fixed with paraformaldehyde and visualized with epifluorescence. Yellow arrows indicate the punctate pattern of GFP-LC3, representative of autophagosome, and nuclei were visualized through DAPI staining. The number of GFP-LC3 punctuate dots/cells was quantitated in 50 GFP+ cells from each group (bottom).
- Figure 5.
Regulation of autophagy proteins in response to combination treatment of Rad001 and propachlor. A, Atg5–Atg12 conjugates and Beclin1 were induced in response to combination treatment for 24 hours. Immunoblotting was conducted as in Fig. 3. B, Beclin 1 mRNA was more significantly upregulated by the combination treatment as shown by qRT-PCR analyses (left). Right, shows that Beclin1 mRNA stability does not increase in response to the combination treatment. C, Beclin1 is critical for the autophagic death induced by the combination treatment. PC3 and C4-2 cells were transfected with control siRNA or siRNA for Beclin1 (right). Cells were treated with combination of Rad001 and propachlor 24 hours after transfection. The cell number was determined with ATP measurement 24 hours after the combination treatment.
- Figure 6.
Combination of Rad001 and propachlor significantly inhibits the PC3 xenograft tumors in mice. A, antitumor activity on PC3 xenografts by various compounds. Mice were administered daily i.p. with Rad001 (1 mg/kg), propachlor (5 mg/kg), or both (6 mice/group). The tumor sizes (left) and body weights (right) were measured every 4 days. Tumors treated with 2 drugs were much smaller than single compound treatment or control. B, electron microscopic analysis of the tumors collected from various treatment groups. Arrows indicate autophagosomes. C, quantitation of the number of autophagosomes in 30 cells from the xenograft tumors. D, immunoblot analysis for LC3 and Beclin1 from the xenograft tumors.
Additional Files
Supplementary Data
Files in this Data Supplement:
- Supplementary Figure Legend - PDF file, 70KB.
- Supplementary Figure 1 - PDF file, 66KB, Mixture-Algebraic estimate analysis of the combination index (CI) values.
- Supplementary Figure 2 - PDF file, 258KB, Cell-cycle distribution histograms.
- Supplementary Table 1 - PDF File, 1123KB, The combination index (CI) values of each mixture treatment calculated with CalcuSyn software for PC3 (A) and C4-2 (B) cells.
Volume 11, Issue 6
