cells after 24 h of treat- ment with7-AAG and sorafenib showed a similar pat- tern with our Western blotting and CI determinations: the percentage decreased with increasing concentration of7-AAG and sorafenib, except at the highest concentra- tion of7-AAG in AsPC-1 cells ( Fig. 4 E and F). DISCUSSION Chemosensitivity is likely to be an inherent, complex phenotype, with genetic polymorphisms, protein 4 ? LIU ET AL.: MULTIPLE sulfanilamide KINASES REGULATE CANCER SENSITIVITY TO7-AAG 5 FIG. 3. Sorafenib antagonizes the effects of7-AAG on multiple kinase pathways. Cell lysates were prepared after 24 h of treatment with7-AAG and sorafenib at the indicated concentrations and separated on polyacrylamide gel. The membrane was probed with the indicated an- tibodies. The treatments had no effect on the level of the co-chaperone molecule CDC37 or its activated form, p-CDC37. However,7-AAG and sorafenib acted antagonistically on the Akt(p-Akt), Wnt (p-GSK3 b ), and MAP kinase (p-ERK1/2) signaling pathways, more markedly so in AsPC-1 cells than in Panc-1 cells.7-AAG and sorafenib had no effect on the total amounts of the indicated proteins, except that they markedly decreased the level of total Akt at the highest concentrations (2 3 IC 50 ) in AsPC-1 cells. A 7-AAG; S sorafenib.

The numbers indicate con- centrations in m M. expression alterations, and purchase sulfanilamide post-translational modia- tions playing signiant roles. In this study, two pancre- atic cancer cell lines, AsPC-1 and Panc-1, provided us with an excellent model to explore the molecular deter- minants of sensitivity to7-AAG, a novel anticancer agent that inhibits the chaperoning function of HSP90 [6] . In the logarithmic growth phase (60%?0% con- ence), AsPC-1 cells were much more sensitive to7- AAG than Panc-1 cells. Many kinases responsible for cell proliferation and survival are client proteins of HSP90. Targeting HSP90 with7-AAG leads to degra- dation of these kinases and cell death [9] . Thus, the dif- ference in the responsiveness of the multiple kinases to HSP90 inhibition might be the basis for the different cy- totoxic effects of7-AAG on AsPC-1 and Panc-1 cells.

Our Western blotting results indeed showed that after 24 h of7-AAG treatment, several key phosphorylated kinases (ERK1/2, MEK1/2, and GSK-3 b ) decreased to much lower levels in AsPC-1 cells than in Panc-1 cells. Growth factors can stimulate Bad phosphorylation, which suppresses cell apoptosis and promotes cell sur- vival [21] . We found that HSP90 inhibition resulted in decreased p-Bad (S112) levels in AsPC-1 cells but in- creased levels in Panc-1 cells. p-Bad (S112) levels may be a predictive factor for the sensitivity of order sulfanilamide pancreatic cancer cells to7-AAG, although the underlying mech- anism for this effect remains to be elucidated. As cytotoxic drugs, HSP90 inhibitors can activate the heat shock response [11, 26] . Our results also revealed that treatment with7-AAG induced expression of the three major HSPs, HSP90, HSP70, and HSP27, espe- cially HSP70 ( Fig. 2 A), which increased to 6.6 3 and 2.4 3 the level without7-AAG treatment in Panc-1 and AsPC-1 cells, respectively. The stronger heat shock response in Panc-1 cells might be attributable to7- AAG resistance because induction of stress response proteins

such as HSP27 and HSP70, by HSP90 inhibi- tion could offset the cytotoxic effects of7-AAG [11] . In the microarray proing of 60 human tumor cell lines (NCI-60), their sensitivity to geldanamycin and its ana- logs displayed negative correlation with mRNA expres- sion levels of P-glycoprotein, suggesting a role of P-glycoprotein in chemoresistance [22] . However, our microarray data of nine pancreatic cancer cell lines did not show any association between7-AAG sensitiv- ity and P-glycoprotein mRNA expression levels. More- over we did not detect any protein expression of P-glycoprotein in AsPC-1 or Panc-1 cells, indicating that P-glycoprotein was not involved in the responsive- ness of AsPC-1 and Panc-1 cells to7-AAG. Mutant p53 pr