Small Molecule Inhibitors of MAPK and PI3K Pathways Enhance MDA-7 Lethality in Renal Cell Carcinoma

Renal cell carcinoma accounted for an estimated 57,760 new cases and estimated 12,980 deaths in the United States in 2009. Current treatment options for systemic renal cell carcinoma yield markedly low response percentages; however, recent cytokine therapy experiments have produced promising results. A novel adenovirus, Ad.5/3-mda-7, has been synthesized to efficiently infect renal cancer cells with the mda-7 gene. This gene encodes for the cytokine MDA-7/IL-24 that has the ability to specifically target transformed cells. Assays performed with this adenovirus resulted in an increased percentage of cell death in renal cancer cells when compared to infection with Ad.5/3-cmv empty vector. Further assays that combined Ad.5/3-mda-7 infection with treatments of small molecule inhibitors increased the percentages of cell death by upregulating JNK and p38 MAPK pathways, downregulating the ERK1/2 MAPK pathway, and downregulating the PI3K pathway. Western blots confirmed upregulation and downregulation of these pathways by probing for key proteins. Renal cancer cells responded best to infection with Ad.5/3-mda-7 and treatment with PD184352, PX866, and Rapamycin. This combinatorial treatment caused a greater percentage of cell death than the sum of the two individual treatments, suggesting a synergistic inhibition of cell growth pathways. These findings suggest that the combination of Ad.5/3-mda-7 and specific small molecule inhibitors has developmental potential as a novel and more efficient treatment option for systemic renal cell carcinoma

Influence of Atrial Function and Mechanical Synchrony on LV Hemodynamic Status in Heart Failure Patients on Resynchronization Therapy

ObjectivesThe aim of this study was to evaluate atrial and ventricular function in patients undergoing cardiac resynchronization therapy (CRT).BackgroundRight atrial pacing (AP) in CRT induces delays in electrical and mechanical activation of the left atrium. The influence of atrial sensing (AS) versus AP on ventricular performance in CRT and the mechanisms underlying the differences between AS and AP in CRT have not been fully elucidated.MethodsFifty-five patients with heart failure undergoing CRT for 9 ± 12.5 months and 22 control subjects without heart failure were enrolled. Conventional and tissue Doppler echocardiography was performed to examine atrial and ventricular mechanics and hemodynamic status.ResultsThe optimal atrioventricular interval was shorter in AS compared with AP mode (126 ± 19 ms vs. 155 ± 20 ms, p < 0.0001). Left ventricular (LV) outflow tract time-velocity integral (22 ± 7 cm vs. 20 ± 7 cm, p < 0.001), diastolic filling period (468 ± 124 ms vs. 380 ± 93 ms, p < 0.001), and global strain (−32 ± 24% vs. −27 ± 22%, p = 0.001) were greater in AS compared with AP mode. Atrial strain was higher in AS compared with AP mode in the right atrium (−28.2 ± 8.6% vs. −22.6 ± 7.6%, p = 0.0007), interatrial septum (−17.1 ± 6.5% vs. −13.2 ± 5.4%, p = 0.002), and left atrium (−16.4 ± 11.0% vs. −13.6 ± 8.5%, p = 0.02). There was no difference in intraventricular dyssynchrony but significantly lower atrial dyssynchrony in AS compared with AP mode (31 ± 19 ms vs. 42 ± 24 ms, p = 0.0002).ConclusionsAS is associated with preserved atrial contractility and atrial synchrony, resulting in optimal LV diastolic filling, stroke volume, and LV systolic mechanics. This pacing mode maximizes LV performance and the hemodynamic benefit of CRT in patients with heart failure

Poly(ADP-Ribose) Polymerase 1 Modulates the Lethality of CHK1 Inhibitors in Carcinoma Cells

Prior studies have demonstrated that inhibition of CHK1 can promote the activation of extracellular signal-regulated kinases 1 and 2 (ERK1/2) and phosphorylation of histone H2AX and that inhibition of poly(ADP-ribose) polymerase 1 (PARP1) can affect growth factor-induced ERK1/2 activation. The present studies were initiated to determine whether CHK1 inhibitors interacted with PARP1 inhibition to facilitate apoptosis. Transient expression of dominant-negative CHK1 raised basal ERK1/2 activity and prevented CHK1 inhibitors from activating ERK1/2. CHK1 inhibitors modestly increased the levels of PARP1 ADP ribosylation and molecular or small-molecule inhibition of PARP1 blocked CHK1 inhibitor-stimulated histone H2AX phosphorylation and activation of ERK1/2. Stimulated histone H2AX phosphorylation was ataxia telangiectasia-mutated protein-dependent. Multiple CHK1 inhibitors interacted in a greater than additive fashion with multiple PARP1 inhibitors to cause transformed cell-killing in short-term viability assays and synergistically killed tumor cells in colony-formation assays. Overexpression of BCL-xL or loss of BAX/BAK function, but not the function of BID, suppressed CHK1 inhibitor + PARP1 inhibitor lethality. Inhibition of BCL-2 family protein function enhanced CHK1 inhibitor + PARP1 inhibitor lethality and restored drug-induced cell-killing in cells overexpressing BCL-xL. Thus, PARP1 plays an important role in regulating the ability of CHK1 inhibitors to activate ERK1/2 and the DNA damage response. An inability of PARP1 to modulate this response results in transformed cell death mediated through the intrinsic apoptosis pathway

Inhibition of MCL-1 in breast cancer cells promotes cell death in vitro and in vivo

The present studies have examined approaches to suppress MCL-1 function in breast cancer cells, as a means to promote tumor cell death. Treatment of breast cancer cells with CDK inhibitors (flavopiridol; roscovitine) enhanced the lethality of the ERBB1 inhibitor lapatinib in a synergistic fashion. CDK inhibitors interacted with lapatinib to reduce MCL-1 expression and overexpression of MCL-1 or knock down of BAX and BAK suppressed drug combination lethality. Lapatinib-mediated inhibition of ERK1/2 and to a lesser extent AKT facilitated CDK inhibitor-induced suppression of MCL-1 levels. Treatment of cells with the BH3 domain/MCL-1 inhibitor obatoclax enhanced the lethality of lapatinib in a synergistic fashion. Knock out of MCL-1 and BCL-XL enhanced lapatinib toxicity to a similar extent as obatoclax and suppressed the ability of obatoclax to promote lapatinib lethality. Pre-treatment of cells with lapatinib or with 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 a synergistic fashion. Overexpression of MCL-1 or knock down of BAX and BAK suppressed the toxic interaction between CDK inhibitors and obatoclax. Obatoclax and lapatinib treatment or obatoclax and CDK inhibitor treatment or lapatinib and CDK inhibitor treatment radiosensitized breast cancer 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 cancer cells more susceptible to BAX/BAK-dependent mitochondrial dysfunction and tumor cell death

Enhancing mda-7/IL-24 therapy in renal carcinoma cells by inhibiting multiple protective signaling pathways using sorafenib and by Ad.5/3 gene delivery

We have determined whether an adenovirus that comprises the tail and shaft domains of a serotype 5 virus and the knob domain of a serotype 3 virus expressing MDA-7/IL-24, Ad.5/3-mda-7, more effectively infects and kills renal carcinoma cells (RCCs) compared to a serotype 5 virus, Ad.5-mda-7. RCCs are a tumor cell type that generally does not express the receptor for the type 5 adenovirus; the coxsakie and adenovirus receptor (CAR). Ad.5/3-mda-7 infected RCCs to a much greater degree than Ad.5-mda-7. MDA-7/IL-24 protein secreted from Ad.5/3-mda-7-infected RCCs induced MDA-7/IL-24 expression and promoted apoptosis in uninfected “bystander” RCCs. MDA-7/IL-24 killed both infected and bystander RCCs via CD95 activation. Knockdown of intracellular MDA-7/IL-24 in uninfected RCCs blocked the lethal effects of conditioned media. Infection of RCC tumors in one flank, with Ad.5/3-mda-7, suppressed growth of infected tumors and reduced the growth rate of uninfected tumors implanted on the opposite flank. The toxicity of the serotype 5/3 recombinant adenovirus to express MDA-7/IL-24 was enhanced by combined molecular or small molecule inhibition of MEK1/2 and PI3K; inhibition of mTOR, PI3K and MEK1/2; or use of the multi-kinase inhibitor sorafenib. In RCCs, combined inhibition of cytoprotective cell signaling pathways enhanced the MDA-7/IL-24-induction of CD95 activation, with greater mitochondrial dysfunction due to loss of MCL-1 and BCL-XL expression and tumor cell death. Treatment of RCC tumors in vivo with sorafenib also enhanced Ad.5/3-mda-7 toxicity and prolonged animal survival. Future combinations of these approaches hold promise for developing a more effective therapy for kidney cancer