whereas the positive control showed strong P-glycoprotein expression. The positive control H460/ TaxR was prepared from a lung cancer cell line, H460, with acquired paclitaxel resistance established by re- peated treatment with Taxol [23] . On the basis of our microarray data for pancreatic cancer cell lines [24] , we used a similar Acetylcysteine pharmacogenomic approach to deter- mine whether there was a correlation between7-AAG sensitivity and mRNA expression levels of P-glycopro- tein in nine pancreatic cancer cell lines (MIAPaCa-2, BxPC-3, AsPC-1, L3.6p1, Hs766T, MPanc96, SU86.86, CFPAC, and Panc-1); we did not identify any associa- tion of P-glycoprotein mRNA levels with7-AAG resis- tance (data not shown).

Panc-1 cells harbor a missense mutation in p53 DNA binding domain and express high levels of mutant p53 3 ? 4 JOURNAL OF SURGICAL RESEARCH: VOL. – , NO. – , – 2011 FIG. 2. Expression of HSP27, HSP70, and HSP90, but not of P-glycoprotein, is induced by the HSP90 inhibitor7-AAG. (A), (C), (D) Cel- lular extracts were prepared and treated with the indicated concentrations of7-AAG for 72 h. Expression levels of the three major HSPs (HSP27, HSP70, and HSP90) were determined by probing with their corresponding Acetylcysteine 616-91-1 antibodies. (A) Compared with b -actin, we found that ex- pression of HSP70 was strongly induced, whereas expression of HSP27 and HSP90 was only moderately induced. Signal intensities were cal- culated for (C) AsPC-1 and (D) Panc-1 cells. (B) Expression of P-glycoprotein (MDR1) was not detectable in AsPC-1 or Panc-1 cells with or without7-AAG treatment. A positive control, TaxR, was included in this Western blot. protein, whereas AsPC-1 cells harbor a frameshift (null) mutation in p53 with no detectable expression of mutant p53 protein (data not shown). We used p53 siRNA and expression vectors and found that mutant p53 protein did not have any impact on the sensitivity of Panc-1 and AsPC-1 cells to7-AAG ( Figs. S1 and S2 , Supplemental Data ). Sorafenib (Nexavar), a Multiple Kinase Inhibitor

         Regulates 17-AAG Sensitivity Sorafenib, originally developed as a Raf kinase inhib- itor, is a multiple kinase inhibitor [25] . We found that sorafenib could paradoxically buy Acetylcysteine up-regulate the levels of multiple phosphorylated kinase substrates in AsPC-1 and Panc-1 cells as determined by Western blotting af- ter 24 h of7-AAG treatment ( Fig. 3 ). Thus, combined treatment with7-AAG and sorafenib had an antago- nistic effect on multiple kinase pathways. We found that levels of p-ERK1/2 (T202/Y204), p-Akt (S473), p-S6 (S235/236), and p-GSK-3 b were elevated after combined treatment with7-AAG and sorafenib for 24 h. The total levels of most of these proteins were un- affected, but total Akt decreased markedly at increas- ing concentrations of7-AAG and sorafenib in AsPC-1 cells. Sorafenib treatment also shifted multiple low- molecular-weight isoforms of B-Raf to high-molecular- weight ones in AsPC-1 cells, with no apparent change in Panc-1 cells ( Fig. 3 ). To further conm our observation that the inherent differences in multiple kinases regulated the sensitivity of AsPC-1 and Panc-1 cells to7-AAG, we extended our Western blot determinations to cytotoxicity assays. AsPC-1 and Panc-1 cells had very similar responses to sorafenib, with IC 50 values of about1.82 m M. At 5 m M, sorafenib seemed to decrease the sensitivity of Panc-1 cells to7-AAG, increasing IC 50 by almost a factor of 5, while not signiantly altering the sensitivity of AsPC-1 cells to7-AAG ( Fig. 4 A and B). To clarify the an- tagonistic   interaction of7-AAG and sorafenib

          we deter- mined the combination index (CI) in AsPC-1 and Panc-1 cells according to Chou and Talalay [19] . Consistent with our results from Western blotting, we found that7-AAG and sorafenib mostly had an antagonistic effect (CI >), except that they acted synergistically (CI <) at high concentrations in AsPC-1 cells ( Fig. 4 C and D). The change in p-HSP90 (T4/5) as a percentage of total HSP90 in AsPC-1 cells and Panc