Figure 1.
Monoagent ISC-4 activity in vitro.
(A) Cell viability assays and calculated EC50 values for indicated cell lines treated with ISC-4 or DMSO (72 hr, n = 3). Effect of ISC-4 treatment on cell cycle profiles of synchronous and asynchronous (B) HCT116 and (C) HT-29 cell lines. Asynchronous cells are indicated by A in the figure. (D) Sub-G1 content of indicated colon cancer cell lines following ISC-4 treatment (0, 1, 2, 4, 8, or 16 µM).
Figure 2.
Combinatorial activity of ISC-4 and FDA-approved cancer therapies.
Cell viability assays in SW480 and RKO colon cancer cell lines treated with ISC-4 (1, 2, or 4 µM) and indicated therapies at putative EC12.5, EC25, and EC50 alone and in combination (n = 3). Doses are provided in Table S1.
Figure 3.
ISC-4 and cetuximab synergize in human colon cancer cells with wild-type KRAS genes independently of 5-FU sensitivity.
(A) Cell viability assays of human colon cancer cell lines treated with ISC-4 and cetuximab at indicated doses for 72 hours (n = 3). (B) Cell viability assay of wild-type and 5-FU-resistant RKO cells treated with 5-FU as indicated for 24 hours (n = 3). (C) 5-FU-resistant RKO cells treated with ISC-4 (2 µM) and cetuximab (1 µg/mL) for 24 hours (n = 3).
Figure 4.
ISC-4 and cetuximab are cooperatively cytotoxic in 5-FU-resistant colon cancer cells.
(A) Cell viability assays of RKO cells treated with ISC-4 (2 µM) and cetuximab (1 µg/mL) alone or in combination for the indicated time period (n = 3). (B) DAPI staining of RKO cells treated as in (A) for 12 hours. White arrows indicate cells with fragmented DNA. (C) Sub-G1 content of RKO cells treated with ISC-4 (2 µM) and cetuximab (1 µg/mL) alone or in combination for 12 hours (n = 3). *P<0.05 compared to all treatment groups by Student's two-tailed t test. (D) Caspase-Glo assay of RKO cells treated with ISC-4 (2 uM) in combination with cetuximab (0, 0.25, 0.5, or 1 µg/mL) at 24 hours post-treatment. Bottom panel shows quantification of ISC-4 (2 uM) and cetuximab (1 µg/mL) (n = 3).
Figure 5.
ISC-4 and cetuximab cooperatively reduce phospho-Akt.
(A) Western blot analysis of RKO cells treated with ISC-4 (2 µM) and cetuximab (1 µg/mL) alone or in combination for 24 hours. Densitometry of phospho-Akt levels are shown above the blot as normalized to Ran as relative to control treatment. (B) Western blot analysis of RKO cells treated with ISC-4 (2 µM) and cetuximab (1 µg/mL) alone or in combination for indicated time periods. Ran is shown as a loading control. (C) Western blot analysis of indicated human colon cancer cell lines following treatment with the combination (Rx) of ISC-4 (2 µM) and cetuximab (1 µg/mL) for 8 hrs. *P<0.05 compared to control.
Figure 6.
The ISC-4 and cetuximab has synergistic anti-tumor effects against advanced 5-FU-resistant colon cancer xenografts.
(A) Relative tumor sizes of 5-FU-resistant RKO xenografts at 4 days post-treatment with a single dose of ISC-4 (3 mg/kg, i.p.), cetuximab (10 mg/kg, i.v.), or the combination (n≥5). Individual tumors were normalized to their baseline size measured on day 0. *P<0.05 compared to all treated groups using Student's two-tailed t test. (B) Hematoxylin and eosin (H&E) staining and TUNEL staining of xenograft tumors harvested 24 hours after treatment. (C) Athymic female nude mice harboring established HT-29 xenograft tumors were treated with ISC-4 (3 mg/kg, i.v.), cetuximab (10 mg/kg, i.v.), the combination, or cetuximab and 5-FU (25 mg/kg, i.v.) once per week starting on day 0 (n≥8). Error bars indicate SEM of replicates. *P<0.05 compared to control.