Figures & data
Figure 1. p-SRC was highly expressed in pancreatic cancer cell lines and can be induced by paclitaxel or gemcitabine.
a: The expression of p-SRC (Y416 and Y527) and total SRC was evaluated in five human pancreatic cancer cells (HPAC, BXPC-3, ASPC-1, PANC-1, and CAPAN-1), three mouse pancreatic cancer cells (22–614-APR, 8–285-APR, and 8–365-APR), and two normal human cells (NHLF and HCMSC). Cells were harvested, and the protein expression was detected by Western blot. GAPDH was served as loading control. b: The relative fold change of protein expression level was normalized to GAPDH and quantified by ImageJ. The relative ratio of protein fold changes (Y416/Y527) was presented in all cell lines (***, p < 0.001 compared with NHLF or HCSMC). c: HPAC human pancreatic cancer cells were treated with paclitaxel (0.1 and 1 µM) or with gemcitabine (0.1 and 1 µM) for 24 h. The protein expression of p-SRC (Y416 and Y527), p-STAT3Y705, p-AKTS473, and p-ERK1/2T202/Y204 were detected by western blots with GAPDH as loading control. d: The relative fold change of protein expression level was quantified by ImageJ. (***, p < 0.001)
Figure 2. Dasatinib shows synergistic effects when combined with paclitaxel or gemcitabine.
a: Human pancreatic cancer cells (HAPC, PANC-1, CAPAN-1, ASPC-1, and BXPC-3) were seeded in 96-well plates at a density of 3,000 cells per well for 24 h and then treated with dasatinib, paclitaxel or dasatinib and paclitaxel combined at the indicated doses for 72 h. Cell viability was performed by MTT. The CI values of all the combination treatments were calculated by CompuSyn software. b: Human pancreatic cancer cells (HAPC, PANC-1, CAPAN-1, ASPC-1, and BXPC-3) were seeded in 96-well plates at a density of 3,000 cells per well for 24 h and then treated with dasatinib, gemcitabine or dasatinib and gemcitabine combined at the indicated doses for 72 h. Cell viability was performed by MTT. The CI values of all the combination treatments were calculated by CompuSyn software. c: Human pancreatic cancer cells (HAPC, PANC-1, and CAPAN-1) were seeded in six-well plates at a density of 50,000 cells per well for 24 h and then treated with paclitaxel or dasatinib and paclitaxel combined at the indicated doses for 2, 4, 6, and 8 days using Trypan blue staining method. d: Human pancreatic cancer cells (HAPC, PANC-1, and CAPAN-1) were seeded in six-well plates at a density of 50,000 cells per well for 24 h and then treated with dasatinib, gemcitabine, or dasatinib and gemcitabine combined at the indicated doses for 2, 4, 6, and 8 days using Trypan blue staining method. (*, p < 0.05; **, p < 0.01; ***, p < 0.001).
Figure 3. Combination of dasatinib with paclitaxel or gemcitabine can greatly inhibit the cell migration of human pancreatic cancer cells.
Wound healing assay was performed in HPAC (a), PANC-1 (b), and BXPC-3 (c) human pancreatic cancer cells. Representative pictures are shown in each cell line. It was conducted by scratching the cells with yellow pipette tip when HPAC, PANC-1, and BXPC-3 cells grew into the monolayer. Then, cells were treated with dasatinib, paclitaxel, and gemcitabine alone and in combination of dasatinib and paclitaxel or dasatinib and gemcitabine. The same concentration of paclitaxel plus gemcitabine and FOLFIRINOX was used to compare the effects at the same time. Cells were then allowed to migrate into the scratched area for 24 h (HPAC and PANC-1 cells) or 6 days (BXPC-3 cells) when the DMSO group heals completely. Red arrows indicate gap of scratched area. The percentage of migrating area in wound healing assay was quantified in HPAC cells. (***, p < 0.001).
Figure 4. Combination of dasatinib with paclitaxel or gemcitabine can greatly inhibit the cell migration of mouse pancreatic cancer cells.
Wound healing assay was performed in 8–285 APR and 8–365 APR mice pancreatic cancer cells. Representative pictures are shown in 8–285 APR (a) and 8–365 APR (b) mice pancreatic cancer cells. It was conducted by scratching the cells with yellow pipette tip when 8–285 APR and 8–365 APR cells grew into the monolayer. Then, cells were treated with dasatinib, paclitaxel, and gemcitabine alone and in combination of dasatinib and paclitaxel or dasatinib and gemcitabine. The same concentration of paclitaxel plus gemcitabine and FOLFIRINOX was used to compare the effects at the same time. Cells were then allowed to migrate into the scratched area for 24 h when the DMSO group heals completely. Red arrows indicate gap of scratched area. The percentage of migrating area in wound healing assay was quantified in 8–285 APR (c) and 8–365 APR cells (d). (***, p < 0.001).
Figure 5. Dasatinib with paclitaxel or with gemcitabine can greatly reduce the cell colony formation ability of human pancreatic cancer cells.
Colony formation assay was conducted in HAPC, PANC-1, CAPAN-1, ASPC-1, and BXPC-3 cells as described in the “Material and methods” section. HAPC, PANC-1, CAPAN-1, ASPC-1, and BXPC-3 human pancreatic cells were treated with dasatinib, paclitaxel, and gemcitabine alone and in combination of dasatinib and paclitaxel or dasatinib and gemcitabine at the indicated doses for 72 h. Same concentration of folfirinox was used simultaneously. After treatment, the same number of cells were seeded and cultured in a drug-free medium for 1–2 weeks. Colonies were fixed by ice-cold methanol and stained by 1% crystal violet.
Figure 6. Combination of dasatinib with paclitaxel or gemcitabine can induce apoptosis and inhibit the activation of SRC/STAT3 pathway.
a: 8,000 cells per well of HAPC, PANC-1 and 8–285-APR cells were treated with 0.1 µM dasatinib, 0.1 µM paclitaxel and a combination of dasatinib and paclitaxel for 6 h. The level of cleaved caspase-3/7 (RFU) was measured using Caspase-3/7 Fluorescence Assay kit. b: HPAC, PANC-1and 8–285 APR cells were treated with dasatinib (0.1 μM), paclitaxel (0.1 μM) alone or in combination for 24 h. The protein expression of p-Src (Y416 and Y527), p-STAT3Y705, p-AKT, p-ERK1/2, and STAT3 were detected by Western blots with GAPDH as loading control. c: 8,000 cells per well of HAPC, PANC-1 and 8–285-APR cells were treated with 0.1 µM dasatinib, 0.1 µM gemcitabine and a combination of dasatinib and gemcitabine for 6 h. The level of cleaved caspase-3/7 (RFU) was measured using Caspase-3/7 Fluorescence Assay kit. d: HPAC, PANC-1and 8–285 APR cells were treated with dasatinib (0.1 μM), gemcitabine (0.1 μM) alone or in combination for 36 h. The protein expression of p-Src (Y416 and Y527), p-STAT3Y705 p-AKTS473, p-ERK1/2T202/Y204, STAT3, ERK, and SRC were detected by Western blots with GAPDH as loading control.
