Supplementary Figures 1-5

Supplementary Figures 1-5

SupplementaryTables 1-2

Triapine Potentiates Platinum-Based Combination Therapy by Disruption of Homologous Recombination Repair

Elena S. Ratner, Yong-Lian Zhu, Philip G. Penketh, Julie Berenblum, Margaret E. Whicker, Pamela H. Huang, Yashang Lee, Kimiko Ishiguro, Rui Zhu, Alan C. Sartorelli, and Z. Ping Lin

Department of Obstetrics and Gynecology, Yale University School of Medicine, New Haven, Connecticut

Fold Sensitisation / CI
Cisplatin (M) / 3.13 / 1.5 / 1.199
6.25 / 1.9 / 1.178
12.5 / 2.1 / 1.169
25 / 3.0 / 0.916

Fold Sensitisation
Triapine (M)
0.25 / 0.5
Cisplatin (M) / 0.31 / 0.9 / 1.8
0.63 / 1.1 / 1.4
1.25 / 1.0 / 1.3
2.5 / 0.9 / 0.8
5 / 0.5 / 0.5

Fig.1. Effects of concurrent and sequential combinations of triapine and cisplatin on MDA-MB231 cells. A. Concurrent treatment with triapine and cisplatin. Cells were continuously treated with 0.25 or 0.5 M triapine in combination with various concentrations of cisplatin. After 14 days, clonogenic survival was determined. Data are means ± S.D. Fold sensitisation was calculated as the ratio of % survival of vehicle-treated cells to % survival of triapine treated cells at each cisplatin concentration. B. Sequential treatment with triapine and cisplatin. Cells were pulse-treated with various concentrations of cisplatin for 1 hr and then treated with 0.5 M triapine continuously in the absence of cisplatin for 14 days. Data are means ± S.D. Fold sensitisation and CI values were determined. *, CI < 1.

Fig.2. Depletion of BRCA2 by siRNA sensitises SKOV-3 cells to olaparib(A) and cisplatin (B). SKOV-3 cells were transfected with BRCA2-siRNA for 24 hr. Cells were trypsinised and plated for 24 hr prior to continuous exposure to various concentrations of olaparib or cisplatin for 14 days. Colonies were stained and clonogenic survival was determined. Data are means  S.D. Confirmation of BRCA2 knockdown was shown in C by western blotting. A moderate degree of sensitisation may be attributable to the transient duration (72-96 hr) of BRCA2 knockdown by siRNA.

Fig. 3.SKOV3-DR-GFP cells were transfected with an empty vector (-SceI) or the I-SceI expression vector (+SceI) for 5 hr, and then treated without and with 0.75 M triapine for 48 hr. Cells were then analysed by flow cytometry. GFP-positive cells were gated and shown in the boxed areas as % of total cells analysed. Representative scatter plots of green fluorescence (y-axis) and side scatter (SSC; x-axis) of 50,000 cells are shown.

Fig.4.Comparison of the growth delay of EOC tumour by doxil, carboplatin, and the doxil-carboplatin combination in nude mice. Nude mice bearing SKOV-3 tumour were treated with single drugs alone or both drugs in combination. The mean of the tumour volumesfrom each treatment group (n=5) was determined. Data are means ± SD (A).The means of body weights of mice were also determined (B). Arrows indicate the days when drugs were given.

Fig. 5.Wild-type p53 supresses HRR activity in SKOV3 cells. SKOV-3-DR-GFP cells were transfected with the empty vector pCMV, the wild-type p53-expressing pCMV-p53, and the mutant p53 (R175H)-expressing pCMV-p53mt plasmids (Clontech, Mountain View, CA). After 48 hr, cells were analysed for GFP-positive populations by flow cytometry.%HRR relative to pCMV-transfected cells (set as 100%) is shown. Asterisks(p<0.05) indicate significant differences from pCMV-transfected cells

Table 1. CI values for simultaneous combinations of triapineand various DNA damaging agents in BG-1 cells (Fig. 2). CI values smaller than 1 are marked in red.

A. Triapine and olaparib combinations.

CI Value
Triapine (M)
0.25 / 0.5 / 0.75
Olaparib (M) / 0.3125 / 0.887 / 0.849 / 0.866
0.625 / 1.251 / 0.961 / 0.869
1.25 / 1.24 / 0.927 / 0.800
2.5 / 1.217 / 0.818 / 0.806
5 / 1.125 / 0.934 / 0.728

B. Triapine and cisplatin combinations.

CI Value
Triapine (M)
0.25 / 0.5 / 0.75
Cisplatin (M) / 0.3125 / 1.300 / 1.171 / 0.875
0.625 / 1.191 / 1.315 / 0.896
1.25 / 1.232 / 1.527 / 1.056
2.5 / 1.435 / 1.683 / 1.394
5 / 1.790 / 2.087 / 2.141

C. Triapine and carboplatin combinations.

CI Value
Triapine (M)
0.25 / 0.5 / 0.75
Carboplatin (M) / 3.125 / 1.851 / 1.158 / 1.055
6.25 / 1.648 / 1.086 / 0.865
12.5 / 1.344 / 1.111 / 0.795
25 / 1.364 / 1.354 / 1.474
50 / 1.522 / 1.590 / 1.409

D. Triapine and doxorubicin combinations.

CI Value
Triapine (M)
0.25 / 0.5 / 0.75
Doxorubicin (nM) / 1.5625 / 1.450 / 1.189 / 0.901
3.125 / 1.403 / 1.192 / 0.963
6.25 / 1.328 / 1.075 / 0.781
12.5 / 1.018 / 1.102 / 1.000
25 / 1.139 / 1.473 / 0.943

Table 2. CI values for sequential combinations of triapineand cisplatin. CI values smaller than 1 are marked in red.

A. BG-1 cells.

Drug Combination / CI Value
Triapine (M) / Cisplatin (M)
0.5 / 3.125 / 0.994
6.25 / 1.062
12.5 / 0.950
25 / 0.987

B. SKOV-3 cells.

Drug Combination / CI Value
Triapine (M) / Cisplatin (M)
0.75 / 6.25 / 0.593
12.5 / 0.482
25 / 0.273