Article Figures & Data
Figures
- Figure 1.
TAI-95 targets the Hec1/Nek2 pathway. A, MDA-MB-231 cells were treated with DMSO (vehicle control) or 1 μmol/L TAI-95 for 3 hours, lysed, and lysates immunoprecipitated by Nek2 antibody to see co-immunoprecipitated Hec1. Control IgG was used as control antibody for immunoprecipitation. Rbt, rabbit; Ms, mouse. B, cells were incubated for the indicated time points with DMSO or TAI-95 and immunoblotted for levels of Hec1 and Nek2 protein expression. Q.Nek2, quantification by ImageJ (inverted mean, ×1,000). C, MDA-MB-468 cells were grown on chamber slides and treated with DMSO or TAI-95 for 48 hours. After fixation, cells were stained with tubulin antibody and DAPI to stain for microtubules and DNA, respectively, and cells (>500) were counted for presence of chromosomal abnormality and expressed as percentage counted. *, P < 0.05; **, P < 0.01, by 2-tailed t test.
- Figure 2.
TAI-95 leads to apoptotic cell death in breast cancer cell lines. A, synchronized cancer cells treated with DMSO, 1 μmol/L TAI-95, or 20 μmol/L topotecan were immunoblotted for the cleavage of PARP. B, cells were treated with 1 μmol/L TAI-95 for 48 hours, stained for Annexin V and PI, and then analyzed with FACS. C, cells were treated with 1 μmol/L TAI-95 for 24 hours, stained for DNA content with PI, and analyzed with FACS, with the exception of MCF7, which was treated with 50 nmol/L TAI-95 for 72 hours. Topo, topotecan.
- Figure 3.
TAI-95 inhibits tumor growth in breast cancer xenograft mouse models. BT474 breast cancer xenograft model in nude mice were used to test the in vivo efficacy of TAI-95. When tumor size reached 200 mm3, a twice daily, 28-day oral dosing treatment of TAI-95 was initiated. Tumor size (A) and body weights (B) were measured. **, P < 0.01; ***, P < 0.001, by 2-tailed t test.
- Figure 4.
TAI-95 inhibits large tumor growth and does not lead to resistance. BT474 (triple-positive) breast cancer xenograft model in nude mice was used to test the in vivo re-dosing efficacy of TAI-95. TAI-95 dosing was re-initiated after 2 weeks of non-dosing period and restarted on day 42 lasting for another 28 days. Vehicle group mice were sacrificed according to animal protocols and tumor volume postulated with linear assumption. A, tumor volume plotted against time. B, starting tumor size was plotted against treatment dose with circles with size and numbers indicating growth rates obtained at the end of the 28-day treatment period. **, P < 0.01; ***, P < 0.001; ****, P < 0.0001, by 2-tailed t test.
- Figure 5.
TAI-95 potency in MDR lines. A, MDR cells treated with TAI-95 for 96 hours and quantitated with MTS assay to generate GI50. MES-SA/Dx5 (uterine sarcoma), NCI/ADR-RES (ovarian carcinoma), and K562R (leukemia) were tested and 2 paclitaxel-resistant breast cancer cell lines are included as reference. B, cells were treated with 50 nmol/L TAI-95 for the indicated time period, collected, RNA extracted, and analyzed with quantitative PCR. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as loading control and mRNA expression is presented as percentage of DMSO (vehicle control). *, P < 0.05; **, P < 0.01; ***, P < 0.001.
- Figure 6.
TAI-95 downregulates MDR expression. A and B, cells were treated with 100 nmol/L TAI-95 for the indicated time period, collected, RNA extracted, and analyzed with quantitative PCR. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as loading control and mRNA expression is presented as percentage of DMSO (vehicle control). C, cells were pretreated with DMSO, 33 nmol/L (lo) or 166 nmol/L (hi) TAI-95 for 6 hours and then treated with the indicated cytotoxic agent for 72 hours and quantitated with MTS assay. Right, the RNA level of Pgp at 6 hours. *, P < 0.05; **, P < 0.01; ***, P < 0.001, by 2-tailed t test.
- Figure 7.
Clinical correlation of TAI-95 target Hec1 in breast cancer. A, representative blots of Hec1 protein expression in breast cancer cell lines. B, average values of quantitated Hec1 protein expression. C, Hec1 gene expression among different molecular subtypes of breast cancer based on GSE20685 dataset. D, one-way cluster analysis of Hec1 (NDC80) and 8 other genes known to associate with Hec1 was performed. A heatmap was drawn according to breast cancer molecular subtypes. Results show differential expression of Hec1 and associated genes among different molecular subtypes of breast cancer.
Tables
- Table 1.
GI50 values of TAI-95 in various breast cancer lines
MDA-MB-231 MDA-MB-468 T47D Hs578T ZR-75-1 MCF7 HCC1954 BT474 ZR-75-30 MDA-MB-453 MDA-MB-361 Paclitaxel 3.00 nmol/L 2.38 nmol/L 3.19 nmol/L 7.99 nmol/L >10 μmol/L >10 μmol/L >10 μmol/L 5.40 nmol/L >10 μmol/L 1.71 nmol/L >10 μmol/L Doxorubicin 213.80 nmol/L 28.27 nmol/L 7.22 nmol/L 423.34 nmol/L 134.87 nmol/L >10 μmol/L 509.20 nmol/L 158.56 nmol/L >10 μmol/L 114.26 nmol/L >10 μmol/L Topotecan 346.73 nmol/L 10.71 nmol/L 9.43 nmol/L 4513 nmol/L 48.39 nmol/L >10 μmol/L 675.90 nmol/L >10 μmol/L 2334 nmol/L 91.39 nmol/L 303.92 nmol/L Gemcitabine 6.44 nmol/L 5.04 nmol/L 10.35 nmol/L >10 μmol/L 5.23 nmol/L >10 μmol/L >10 μmol/L >10 μmol/L >10 μmol/L ND >50 μmol/L Irinotecan >10 μmol/L 1997 nmol/L 724.70 nmol/L >50 μmol/L >10 μmol/L >10 μmol/L >10 μmol/L >50 μmol/L >10 μmol/L ND >50 μmol/L Gefinitib >10 μmol/L 9093 nmol/L >10 μmol/L >10 μmol/L >10 μmol/L >10 μmol/L >10 μmol/L 774.00 nmol/L 4820 nmol/L ND >10 μmol/L Cisplatin >10 μmol/L >10 μmol/L >50 μmol/L >10 μmol/L >50 μmol/L >50 μmol/L >10 μmol/L >50 μmol/L >50 μmol/L ND >50 μmol/L Cyclophosphamide >500 μmol/L >500 μmol/L >500 μmol/L >50 μmol/L >50 μmol/L >50 μmol/L >50 μmol/L >50 μmol/L >50 μmol/L ND >50 μmol/L KX01 43.41 nmol/L 26.50 nmol/L 51.98 nmol/L 37.79 nmol/L >10 μmol/L >10 μmol/L >10 μmol/L >10 μmol/L >10 μmol/L 53.47 nmol/L 32.18 nmol/L Sorafenib 3760 nmol/L 3172 nmol/L 1730 nmol/L 7252 nmol/L 4541 nmol/L >10 μmol/L 4661 nmol/L 4638 nmol/L 4074 nmol/L 3461 nmol/L 4441 nmol/L Sunitinib >10 μmol/L >10 μmol/L >10 μmol/L >10 μmol/L >10 μmol/L >10 μmol/L >10 μmol/L >10 μmol/L >10 μmol/L ND >10 μmol/L TAI-95 16.48 nmol/L 22.21 nmol/L 14.29 nmol/L 43.39 nmol/L 52.35 nmol/L 39.17 nmol/L 25.46 nmol/L 73.65 nmol/L 25.47 nmol/L > 10 μmol/L > 10 μmol/L Abbreviation: ND, not determined.
Supplementary Data
Files in this Data Supplement:
- Supplementary Materials and Methods, Supplementary Table 1, and Supplementary Figure Legends - PDF - 126K, Includes the methods for preparation of TAI-95, drug-drug synergy experiments, and preliminary subacute toxicology, Table S1: Combination index of TAI-95 with anticancer agents, and supplementary figure legends.
- Supplementary Figures 1 through 6 - PDF - 1333K, Figure S1. Summary of structural modifications. Figure S2. Hec1 localization. Figure S3. Activation of apoptosis. Figure S4. Subacute toxicity study: histological sections. Figure S5. Subacute toxicity study: organ weight and blood indices. Figure S6. Additional breast cancer xenograft mouse models.
Volume 13, Issue 6
