Figure 4

α-TEA cooperates with doxorubicin or cisplatin to upregulate pro-apoptotic and downregulate anti-apoptotic mRNAs and proteins. α-TEA cooperates with doxorubicin (DOXO) or cisplatin (CDDP) to upregulate mRNAs and proteins of the pro-apoptotic mediators death receptor 5 (DR5), Fas, Bax, and Noxa, and downregulate anti-apoptotic Bcl-2 mRNA and protein, all of which are downstream targets of p73. MDA-MB-231 and BT-20 cells were treated with DOXO or CDDP alone or in combination with α-TEA for 24 hours. mRNA levels of DR5, Fas, Bax, Noxa and Bcl-2 were determined by RT-PCR with β-actin serving as loading control (a), (b). Protein levels of DR5, Fas, Bax, Noxa and Bcl-2 were determined by western blot analyses with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) serving as loading control (c), (d). The same treated samples as Figure 3d were used to detect the effect of siRNA to p73 on the combination-induced increase in protein levels of DR5, Fas, Bax, Noxa and decrease in Bcl-2 by western blot analyses with GAPDH as loading control (e). MDA-MB-231 cells were transfected with DR5 siRNA or control siRNA for 2 days and treated with combinations of α-TEA + DOXO or α-TEA + CDDP for 24 hours. Western blot analyses were used to determine the effect of siRNA to DR5 on combination-induced poly(ADP-ribose) polymerase (PARP) cleavage and to verify the knockdown efficiency of DR5 (f). Data representative of at least two independent experiments.