SEARCH

SEARCH BY CITATION

Additional Supporting Information may be found in the online version of this article.

FilenameFormatSizeDescription
IJC_25044_sm_SuppFig1.tif776KSupporting Information Figure 1. Continuous treatment with ursolic acid caused cytotoxicity to ASTC-a-1 and normal cells. Cells were treated continuously with 100μM, 10μM, 1μM ursolic acid for designated times and then cells viability was assayed. Results represent one of three replicates.
IJC_25044_sm_SuppFig2.tif1803KSupporting Information Figure 2. Treatment cells with ursolic acid first and then removing ursolic acid at appropriate time did not cause obvious cytotoxicity to cells. ASTC-a-1 cells were first treated with 100μM ursolic acid for 8h or 10μM, 1μM ursolic acid for 48h. After removal of ursolic acid, cells were cultured in fresh medium for various durations (12h, 1day, 2days, 3days, and 5days), and then cell viability was assayed. Results represent one of three replicates.
IJC_25044_sm_SuppFig3.tif664KSupporting Information Figure 3. Potentiation of Taxol or cisplatin induced cytotoxicity by ursolic acid in vivo. Ursolic acid drastically reduced the required dosage of the chemotherapeutic-agents to achieve identical biological endpoints. At 6 days after dosing, the combination treatment resulted in tumor regression, whereas the corresponding single drug treatments resulted in minimal tumor growth suppression. Long term data shows that oral administration of ursolic acid (500mg/kg daily for 30days in mouse model) resulted in no distinctive effect on body weight, blood, cardiogram, liver, and kidney functions, and no pathology changes for important organs (41). Thus, the dose of 500mg/kg ursolic acid (oral) were used in our experiments in vivo. Athymic nude mice (n=6) were injected s.c. with tumor cells (4×106 cells per animals). When mean tumor volumes were about 400mm3, treatment were initiated. The size of tumors used in experiments mimics severe minimal residual tumor after surgical debulking. Mice were treated with taxol (6 mg/kg, 2 mg/kg), or cisplatin (5 mg/kg, 2.5 mg/kg) alone, or ursolic acid (500mg/kg) combined with either taxol (2 mg/kg) or cisplatin (2.5 mg/kg). For these experiments, Taxol was intraperitoneally administered twice weekly for 3 weeks; cisplatin was intraperitoneally administered once weekly for 3 weeks; ursolic acid were orally administered. In combination group, treatment with Taxol or cisplatin were given after treatment with ursolic acid for 1day. Tumor size was measured every 3 to 4 days. Tumor volumes were calculated from the formula (l×w2)/2, and data were expressed relative to the initial tumor volume [(T/T0)×100].
IJC_25044_sm_SuppFig4.tif678KSupporting Information Figure 4. Transcriptional activation of NF-κB induced by TNFα or chemotherapeutic-agents was completely inhibited by ursolic acid and partially inhibited by wortmannin (Akt inhibitor). ASTC-a-1 cells stably expressing pNF-κB-Luc were first treated with 100μM ursolic acid for 8h. After removal of ursolic acid, cells were treated with TNFα (10ng/ml) or chemotherapeutic-agents (1μM Taxol or 20μM cisplatin). Treatment with TNFα, Taxol, or cisplatin served as comparisons. 1μM wortmannin was added 30min earlier before chemotherapy treatment. Results represent one of three replicates.
IJC_25044_sm_SuppFig5.tif1755KSupporting Information Figure 5. Representative images of immunofluorescence assay of NF-κB p65 nuclear translocation in ursolic acid-chemo treatment (at the concentrations studied). Similar to the results of western blot and luciferase reporter assay, the chemotherapeutic-agents induced NF-κB p65 nuclear translocation were completely inhibited by 10-100μM ursolic acid and partially by 1μM ursolic acid. After removal of ursolic acid, the inhibitive effect of ursolic acid on NF-κB still remained at 48h and reversed completely reversed at 72h. ASTC-a-1cells were cultured on coverslips. Before immunofluorescence assay, cells were first treated with various concentrations of ursolic acid (as indicated in Materials and Method), and then exposed to chemotherapeutic-agents for 1h. Subsequently, cells were fixed in 4℅ formaldehyde for 15 min. After washing, cells were stained with 1:50 antibody against NF-κB p65 overnight at 480C and with secondary antibody of Alexa Fluor 680 goat anti-mouse IgG for 60 min. Coverslips were viewed by LSM microscope. Results represent one of three replicates.
IJC_25044_sm_SuppFig6.tif650KSupporting Information Figure 6. Effects of relatively low doses ursolic acid-chemo treatment on transcriptional activation of NF-κB. Prolonged treatment with relatively low doses of ursolic acid can also suppress the transcriptional activation of NF-κB induced by low doses chemotherapeutic-agents (10nM Taxol or 2μM cisplatin). ASTC-a-1 cells stably expressing pNF-κB-Luc were first treated with 100μM ursolic acid for 8h or 10μM, 1μM ursolic acid for 48h and then ursolic acid was removed. Subsequently, the treated cells were exposed to the chemotherapeutic-agents alone. Treatment with Taxol or cisplatin served as comparisons. Results represent one of three replicates.
IJC_25044_sm_SuppFig7.tif717KSupporting Information Figure 7. Treatment with ursolic acid first and then removing ursolic acid makes ASTC-a-1 cells insensitive to chemotherapeutic-agents induced transcriptional activation of NF-κB for at least 48h. Cells stably expressing pNF-κB-Luc were first treated with 100μM ursolic acid for 8h or 10μM ursolic acid for 48h (The doses make a complete inhibition of NF-κB). After removal of ursolic acid, cells were cultured in fresh medium for various durations (48h, 60h, 72h) and then treated with chemotherapeutic-agents alone (10nM Taxol or 2μM cisplatin). Treatment with Taxol or cisplatin served as comparisons. Results represent one of three replicates.

Please note: Wiley Blackwell is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.