eriments. RESULTS Effects of HSP90 Inhibition on Tumor Cell Survival and Multiple Kinase Pathways To determine the responses of two human pancreatic cancer cell lines, AsPC-1 and Panc-1, to7-AAG, we used MTT assays. The sensitivity of Panc-1 cells was substantially dependent on the conence of the cell cultures, so Panc-1 cells exhibited  Linezolid conence- dependent resistance (CDR) ( Fig. A). AsPC-1 cells, however, did not exhibit substantial CDR. To compare the sensitivity of the two cell lines to7-AAG, we grew the cells to 60%?0% conence before plating them for cytotoxicity assays. We found that Panc-1 cells were much more resistant to7-AAG than AsPC-1 cells were, consistent with the results of a previous study [20] ; Panc-1 and AsPC-1 cells had IC 50 values of 3.18 and 0.12 m M, respectively ( Fig. B).

The different responses of AsPC-1 and Panc-1 cells to7-AAG may be due to the different sensitivities of 2 ? LIU ET AL.: MULTIPLE KINASES REGULATE CANCER SENSITIVITY TO7-AAG 3 FIG.. Multiple kinase signaling pathways are more sensitive to7-AAG in AsPC-1 cells than in Panc-1 cells. (A), (B) We used MTT cell survival assays to determine the cytotoxic effect of7-AAG on AsPC-1 and Panc-1 cells after 72 h of treatment. Each experiment was performed in triplicate. Bars, SD. (A) Panc-1 cells  buy Linezolid exhibited CDR. (B) Sensitivity was determined in the logarithmic growth phase (60%?0% conence), and IC 50 values were 0.12 m M for AsPC-1 cells and 3.18 m M for Panc-1 cells. (C) Cell lysates were prepared after 24 h of treatment with7-AAG at different concentrations and separated on polyacrylamide gel. The membrane was probed with the indicated antibodies. Lanes?, AsPC-1 cells treated

We determined the sensitivities of these kinases to HSP90 inhibition by Western blotting after 24 h of treatment with vary- ing concentrations of7-AAG. Although the total amounts of the kinases were unaffected, the levels of phosphorylated ERK1/2 (p-ERK1/2) and p-MEK1/2, in the MAP kinase pathway, were much more sensitive to7-AAG in AsPC-1 cells than in Panc-1 cells. Simi- larly, levels of p-S6 ribosomal protein and phosphory- lated glycogen synthase kinase-3 b (p-GSK-3 b ) were much more sensitive in AsPC-1 cells than in Panc-1 cells. In contrast, levels of p-Bad (S112) changed in op- posite  purchase Linezolid directions in the two cell lines with increasing7-AAG concentration, decreasing in AsPC-1 cells and increasing in Panc-1 cells, whereas p-Bad (S136) was almost undetectable in both AsPC-1 and Panc-1 cells ( Fig. C). p-Bad is sequestered in the cytosol by binding to4-3-3 to promote cell survival [21] ; the differential changes of p-Bad (S112) levels after7-AAG treatments might be responsible for the different cytotoxic effects of HSP90 inhibition in AsPC-1 and Panc-1 cells. In addi- tion, CDK4 levels were more sensitive to7-AAG in AsPC-1 cells than in Panc-1 cells (data not shown). Expression of Heat Shock Proteins, not P-Glycoprotein, Is Induced by7-AAG Treatment Because7-AAG inhibits HSP90 chaperoning func- tion [2] , we determined the expression levels of various HSPs in AsPC-1 and Panc-1 cells.

We found that the expression levels of the three major HSPs, HSP27, HSP70, and HSP90, were comparable in AsPC-1 and Panc-1 cells without7-AAG treatment. After 72 h of7-AAG treatment, expression of HSP70 was strongly induced, whereas expression of HSP27 and HSP90 was only moderately induced ( Fig. 2 A). Induction of HSP70  inheritance  expression was more pronounced in Panc-1 cells (6.6 3 the level without7-AAG treatment) than in AsPC-1 cells (2.4 3 the untreated level) ( Fig. 2 C and D). P-glycoprotein has been reported to mediate the re- sistance of cancer cells to7-AAG [11,2, 22] . However, after 72 h of treatment with7-AAG at indicated con- centrations, P-glycoprotein was not detectable in AsPC-1 or Panc-1 cells with Western blotting ( Fig.