Fig 1.
Determining effective treatment dose and duration for commercially-available and PDX-derived colorectal cell lines.
(A) Treatment of HT29 and HCT116 cells with FND-4b at varying concentrations for 48h and 72h demonstrated maximal AMPK activation with 10μM FND-4b at 48h. (B) Treatment of HT29, HCT116, LS174T, and DLD1 with 10μM FND-4b for 24h or 48h duration indicated optimal pAMPKα activity at 24h for all cells except DLD1. (C) Treatment of PI3KCA mut/wt DLD1 cells with 10μM FND-4b for 48 h resulted in similar levels of AMPK activation. (D) Treatment with 10μM FND-4b resulted in equivalent AMPK activation at 24h and 48h duration in all four PDX-derived CRC cell lines. Lowest effective treatment dose was 10μM FND-4b for 24h duration. β-actin was used as the loading control for all blots. The images are representative of three independent experiments.
Fig 2.
AMPK activation in commercial & PDX-derived CRC cell lines treatment combinations.
Western blot analysis indicated that AMPK activation was increased in commercially-available CRC cell lines (A) and PDX-derived cell lines (B) after monotreatment or combination treatment with FND-4b (10μM), PI-103 (5μM), or SN-38 (100nM) for 24 h compared to the untreated control. β-actin was used as the loading control. The images are representative of three independent experiments.
Fig 3.
Cell proliferation of commercial CRC cell lines treatment combinations.
(A) Western blot analysis of commercially-available CRC cell lines treated with 10μM FND-4b, 5μM PI-103, and 100nM SN-38, alone and in combination versus untreated control (media alone) for 24h duration. β-actin was used as a loading control. The images are representative of three independent experiments. In all CRC cell lines, FND-4b mono- and dual-treatment resulted in decreased cyclin D1 expression and—with the exception of DLD1—increased pAKT expression compared to untreated control. (B) SRB Cytotoxicity Assays of commercially-available CRC cell lines treated with 10μM FND-4b, 5μM PI-103, and 100nM SN-38, alone and in combination for 48h duration. Graphic representations are the mean ± SD plotted as a percentage relative to untreated control; each measurement was performed with 5 replicates. *p<0.0001 vs. control and #p<0.001 vs. FND-4b alone.
Fig 4.
Apoptosis of commercial CRC cell lines treatment combinations.
(A) Western blot analysis of commercially-available CRC cell lines treated with 10μM FND-4b, 5μM PI-103, and 100nM SN-38, alone and in combination versus untreated control (media alone) for 24h duration. PARP cleavage is indicated by an arrow; β-actin was used as a loading control. The images are representative of three independent experiments. In all CRC cell lines, PARP cleavage was increased as a result of FND-4b treatment compared to untreated control. (B) DNA fragmentation measured by Cell Death ELISA Assays of commercially-available CRC cell lines treated with 10μM FND-4b, 5μM PI-103, and 100nM SN-38, alone and in combination for 48h duration. Graphic representations are the mean ± SD of DNA fragmentation plotted as absorbance (Au); each measurement was performed with 3 replicates. *p<0.01 vs. control and #p<0.01 vs. FND-4b alone.
Fig 5.
Cell proliferation of PDX-derived CRC cell lines treatment combinations.
(A) Western blot analysis of PDX-derived CRC cell lines treated with 10μM FND-4b, 5μM PI-103, and 100nM SN-38, alone and in combination versus untreated control (media alone) for 24h duration. β-actin was used as a loading control. The images are representative of three independent experiments. In all CRC cell lines mono- or dual-treatment with FND-4b resulted in decreased cyclin D1 expression. (B) SRB Cytotoxicity Assays of PDX-derived CRC cell lines treated with 10μM FND-4b, 5μM PI-103, and 100nM SN-38, alone and in combination for 144h duration. Graphic representations are the mean ± SD plotted as a percentage relative to untreated control; each measurement was performed with 5 replicates. *p<0.0001 vs. control, #p<0.01 vs. FND-4b alone, and +p<0.05 vs. SN-38 alone.
Fig 6.
Apoptosis of PDX-derived CRC cell lines treatment combinations.
(A) Western blot analysis of PDX-derived CRC cell lines treated with 10μM FND-4b, 5μM PI-103, and 100nM SN-38, alone and in combination versus untreated control (media alone) for 24h duration. PARP cleavage is indicated by an arrow; β-actin was used as a loading control. The images are representative of three independent experiments. In all CRC cell lines, PARP cleavage was increased as a result of FND-4b treatment compared to untreated control. (B) DNA fragmentation measured by Cell Death ELISA Assays of PDX-derived CRC cell lines treated with 10μM FND-4b, 5μM PI-103, and 100nM SN-38, alone and in combination for 48h duration. Graphic representations are the mean ± SD of DNA fragmentation plotted as absorbance (Au); each measurement was performed with 3 replicates. *p<0.05 vs. control and #p<0.01 vs. FND-4b alone.