The activity of BAK and BAX was determined after immunoprecipitation of the conformationally active BAX and BAK proteins. obatoclax enhanced basal levels of BAX and BAK activity and further enhanced drug combination toxicity. In vivo tumor growth data in xenograft and syngeneic model systems confirmed our in vitro findings. Treatment of cells with CDK inhibitors enhanced the lethality of obatoclax in IPI-549 a synergistic fashion. Overexpression of MCL-1 or knock down of BAX and BAK suppressed the harmful conversation between CDK inhibitors and obatoclax. Obatoclax and lapatinib treatment or obatoclax and CDK inhibitor treatment or lapatinib and CDK inhibitor treatment radiosensitized breast malignancy cells. Lapatinib and obatoclax interacted to suppress mammary tumor growth in vivo. Collectively our data demonstrate that manipulation of MCL-1 protein expression by CDK inhibition or inhibition of MCL-1 sequestering function by Obatoclax renders breast malignancy cells more susceptible to BAX/BAK-dependent mitochondrial dysfunction and tumor cell death. and AIF, leading ultimately to apoptosis. Tumor cells utilize a quantity of Rabbit Polyclonal to MAP2K3 mechanisms to maintain viability, including loss of death receptor expression, e.g., CD95, by losing expression of pro-apoptotic BH3 domain name proteins, e.g., BAX or by increasing expression of anti-apoptotic BCL-2 family members, e.g., MCL-1.24,25 In the case of protective BCL-2 family proteins, several clinically relevant small molecule inhibitors have been developed that specifically bind to the BCL-2 family protein, without altering expression of the protein and that block the binding of pro-apoptotic BH3 domain name proteins, e.g., GX15-070 (Obatoclax).26,27 The drug-induced dissociation of BCL-2 protein from toxic BH3 domain name protein results in greater levels of free BH3 domain name protein that will facilitate mitochondrial dysfunction and promote the toxicity of other therapeutic agents.28,29 The present studies decided whether inhibition of BCL-2 family function using either CDK inhibitors to reduce protein expression or using Obatoclax to inhibit BH3 domain function, could promote tumor cell death. Results The impact of combined exposure of breast malignancy cells to the CDK inhibitor flavopiridol and the ERBB1/ERBB2 inhibitor lapatinib was first investigated. In short-term cell viability assays simultaneous combined exposure of breast malignancy cells to flavopiridol and lapatinib resulted in a greater than additive induction of short-term cell killing compared to either drug individually, which was synergistic as determined by Median Dose Effect analyses with Combination Index (CI) values consistently less than 1.00 (Fig. 1A and Table 1). These findings correlated with dephosphorylation of ERBB1, ERK1/2 and AKT. Parallel studies with another CDK inhibitor, roscovitine, generated data that was very similar to that generated using flavopiridol (Fig. 1B). Constitutive activation of MEK1 and of MEK1 and AKT, protected breast malignancy cells from flavopiridol + lapatinib lethality that correlated with increased MCL-1 expression (Fig. 1C). Overexpression of either BCL-XL or of dominant unfavorable caspase 9, but not c-FLIP-s, suppressed drug lethality (Fig. 1D). Lapatinib enhanced the rate of flavopiridol-induced MCL-1 depletion and overexpression of MCL-1 guarded cells from flavopiridol + lapatinib lethality (Fig. 1E). Treatment of cells with lapatinib and flavopiridol enhanced BAX and BAK activation and knock down of BAX + BAK suppressed flavopiridol IPI-549 + lapatinib lethality (Fig. 1F). In colon cancer cells that were generated to be lapatinib resistant and that we had exhibited was due to increased basal levels of MCL-1, flavopiridol partially circumvented lapatinib resistance (Fig. 1G). Open in a separate window Physique 1ACD Flavopiridol and lapatinib interact in a greater than additive manner to promote mammary tumor cell death in vitro. (A) MCF7, BT474 and SKBR3 were plated as in methods and 24 h after plating concurrently treated with vehicle control (VEH, IPI-549 DMSO), flavopiridol (FP, 50 nM) and/or lapatinib (Lap, 1 M). Cell viability was decided in triplicate 48 h after drug exposure using trypan blue exclusion assays (SEM, n = 3; *p 0.05 greater than additive reduction in viability for the.