A, BxPC-3 and MIAPaCa-2 cells were transfected either with OGX-011 (1200nM) and then challenged with gemcitabine dose of 1.0 uM at 24 h. FACS analysis demonstrating that OGX-011 enhanced gemcitabine toxicity in both of the cells. B, Comparative viability of MIAPaCa-2 cells and BxPC-3 cells before and after sCLU sliencing. Cells were cultured in 96-well plates, then transfected either with OGX-011. Twenty-four hours after last transfection, cells were treated with gemcitabine. Seventy-two hours after drug addition
,cell viability was estimated. The data shown are representative of three independent experiments Angiogenesis inhibitor (*P < 0.05). On the other hand, cellular viability was studied under experimental conditions similar to this described above. Figure 2B shows significantly less viability of MIAPaCa-2 cells and BxPC-3 cells pre-treated with 1200nM OGX-011(* P < 0.05). Together, the aforementioned data indicate that silencing sCLU by OGX-011 enhanced gemcitabine toxicity in the pancreatic cancer cells. Control oligodeoxynucleotide did not have obvious effect on apoptosis or growth in both cells Selleck Staurosporine (data not shown). ERK inhibitor PD98059 inactivates ERK1/2 in untreated and gemcitabine-treated pancreatic cancer cells Studies were then performed to assess the effects of
gemcitabine on ERK1/2 activation in BxPC-3 and MIAPaCa-2 cells. Exposure to 0.5-1.0 μM gemcitabine (18 hr) induced ERK1/2 activation in BxPC-3 cells (Figure 3A).In MIAPaCa-2 cells, 0.5-1.0 μM gemcitabine treatment did not affact ERK1/2 activation (Figure 3A). However, co-administration of the 5 μM ERK inhibitor PD98059 essentially abrogated expression of pERK1/2 in both untreated and gemcitabine -treated BxPC-3(Figure 3B) and MIAPaCa-2 cells (Figure 3B). These findings indicate that in breast cancer
cells, 5 μM ERK inhibitor PD98059 essentially abrogate basal ERK1/2 activation as well as gemcitabine -mediated ERK1/2 activation. Figure 3 ERK inhibitor PD98059 inactivate ERK1/2 in untreated and gemcitabine-treated breast cancer cells. A, BxPC-3 and MIAPaCa-2 cells were exposed to the indicated concentrations of gemcitabine for 18 acetylcholine hr. The cells were then lysed and subjected to WB analysis to monitor pERK1/2 (Thr42/Tyr44) expression as described in Materials and Methods. B, BxPC-3 and MIAPaCa-2 cells were exposed (18 hours) to either 5 μM PD98059, 0.5-1.0 μM of gemcitabine, or the combination, after which proteins were prepared and subjected to WB as described above to monitor pERK1/2 expression. For (A) and (B), lanes were loaded with 25 μg of protein; blots were then stripped and re-probed with GAPDH to ensure equivalent loading and transfer. Representative results are shown; two additional studies yielded equivalent results.