Clobenpropit enhances anti-tumor effect of gemcitabine in pancreatic cancer

Figure 1 Western blotting of histamine receptors in pancreatic cancer cells. Cellular extracts of Panc-1, MiaPaCa-2 and AsPC-1 were separated by SDS-PAGE, transblotted onto nitrocellulose membranes and detected using anti-H₄ receptor antibodies. Pancreatic cancer cell lines were treated with different concentrations of clobenpropit and/or gemcitabine, and Western blotting was performed. The specific antibody to H₄ receptor showed immunoreactivity mainly as a band at 70000 and 43000 Da. Expression of H₄ receptor was still present after administration of clobenpropit alone (A) or combination with gemcitabine (B).

Figure 2 Effect of clobenpropit on cell migration. Clobenpropit inhibits the migration of Panc-1 (A), MiaPaCa-2 (B) and AsPC-1 (C) in wound healing assay. Wound healing range was inversely correlated with clobenpropit concentration in MiaPaCa-2 and AsPC-1.^aP < 0.05, ^bP < 0.01 vs control.

Figure 3 Effect of clobenpropit on pancreatic cancer cell proliferation was evaluated by MTS assay. A: Panc-1, B: AsPC-1. Clobenpropit showed no cytotoxicity in pancreatic cancer cells.

Figure 4 Migration of pancreatic cancer cells was inhibited after gemcitabine and clobenpropit combination in wound healing assay. The concentrations of gemcitabine in Panc-1 (A) and MiaPaCa-2 (B) were 5 and 15 μmol/L, respectively. The concentration of clobenpropit was 50 μmol/L. G: Gemcitabine; C: Clobenpropit.

Figure 5 Changes of epithelial and mesenchymal markers of pancreatic cancer cells by clobenpropit. Real-time polymerase chain reaction shows that E-cadherin was up-regulated (A), whereas MMP-9 and vimentin were down-regulated (B, C) in pancreatic cancer cells after treatment with different concentrations of clobenpropit.

Figure 6 Enhanced apoptosis of pancreatic cancer cells by gemcitabine and clobenpropit combination treatment. Clobenpropit enhanced apoptotic cell death in combination of gemcitabine in human pancreatic cancer cells. The percentage of apoptotic cells was determined by fluorescein isothiocyanate-labeled annexin V assay followed by flow cytometry. Statistically significant differences (^aP < 0.05 vs control group) of the combination treatment of gemcitabine (5 μmol/L) and clobenpropit (50 μmol/L) compared with control in Panc-1 (A), MiaPaCa-2 (B) and AsPC-1 (C). G: Gemcitabine; C: Clobenpropit.

Figure 7 Inhibition of tumor growth in Panc-1 xenograft mouse by gemcitabine and clobenpropit combination treatment. Body weight (A) and tumor volume (B) curves for Panc-1 xenograft mouse model with administration of vehicle (control), gemcitabine, clobenpropit or their combination. There was no significant difference of body weight between the groups. Tumor volume of gemcitabine and clobenpropit combination therapy group was significant lower than control and other treatment groups. Tumor bearing mice and excised tumor of each treatment group (C). G: Gemcitabine; C: Clobenpropit. ^aP < 0.05, ^bP < 0.01 vs control.

Figure 8 Effect of gemcitabine and clobenpropit combination treatment in Panc-1 xenograft mouse. A: Real-time polymerase chain reaction shows increased E-cadherin expression after clobenpropit treatment compared with gemcitabine alone; B: Immunohistochemical staining shows up-regulation of E-cadherin in gemcitabine and clobenpropit combination group. Apoptotic index calculated with TUNEL staining shows increased apoptosis in gemcitabine and clobenpropit combination group; C: Pathological evaluation of tumor tissue determined by hematoxylin and eosin staining, TUNEL staining and immunohistochemistry of E-cadherin and vimentin; D: E-cadherin was also increased in clobenpropit alone and combination group by Western blotting, whereas Zeb1, the repressor of E-cadherin, was decreased in combination group. G: Gemcitabine; C: Clobenpropit. ^aP < 0.05, ^bP < 0.01 vs control.