1, 9-Pyrazoloanthrones Downregulate HIF-1α and Sensitize Cancer Cells to Cetuximab-Mediated Anti-EGFR Therapy
Figure 1
1, 9 PA downregulates HIF-1α independent of its JNK inhibitory function.
(A) Temporal order and correlation of 1, 9 PA-induced HIF-1α downregulation and JNK inhibition, and the treatment-induced compensatory activation of Akt and Erk. A431 cells were treated with 5 µM 1, 9 PA in 0.5% FBS culture medium for the indicated time intervals. Cell lysates were then prepared for Western blotting with the antibodies shown. (B) Dose-dependent effects of 1, 9 PA on downregulating HIF-1α, inhibiting JNK, and activating Erk. A431 cells were exposed to increasing concentrations of 1, 9 PA for 16 h in 0.5% FBS culture medium at 37°C. Cell lysates were then prepared for Western blotting with the antibodies shown. (C) Independence of 1, 9 PA-induced HIF-1α downregulation from its JNK inhibitory function. A431 cells were treated with increasing concentrations of 1, 9 PA or 1-methyl-1, 9 PA for 1 h in 0.5% FBS culture medium. Cell lysates were then prepared for Western blotting with the antibodies shown. (D) No changes in HIF-1α level after activation or inhibition of JNK. A431 cells wert transiently transfected with a control vector or one of the constructs containing a constitutively active SEK1 S220E/T224D mutant (SEK1-CA), a dominant-negative SEK1 K129R mutant (SEK1-DN), or a dominant-negative MEKK1 K432M mutant (MEKK1-DN) overnight. Cell lysates were then prepared for Western blotting with the antibodies shown. (E) Compensatory activation of Erk after HIF-1α silencing in comparison with treatment by 1, 9 PA or 1-methyl-1, 9 PA. A431 cells were subjected to knockdown of HIF-1α with specific or control siRNA, or treated with 10 µM 1, 9 PA or 1-methyl-1, 9 PA for 16 h. Cell lysates were then prepared for Western blotting with the antibodies shown. (F) Downregulation of HIF-1α by 1, 9 PA in various cancer cell lines. Indicated cell lines were exposed to 10 or 40 µM 1, 9 PA for 1 h in 0.5% FBS culture medium. Cell lysates were then prepared for Western blotting with the antibodies shown.