Pharmacologic inhibition of NF-kB leads to suppression of reovirus induced cell death in BrCa cells.

<p>MCF7 and HTB 133 cells were pre-incubated with either the NF-kB specific inhibitors CAPE (20 μM), or the proteasome inhibitor ALLN (10 μM), for 3 hours prior to reovirus infection. Cell death was assessed 24, and 48h via the WST assay (N = 5, ± SD). (*), significantly different.</p

Reovirus upregulates NF-kB activity in BrCa cells.

<p>(A) NF-kB p65 mRNA expression is induced in reovirus infected BrCa cells. HTB133 and MCF7 cells were infected with 40 MOI of LV or DV for 12 and 24 hours. Total RNA extracted was assessed for NF-kB p65 and 18S gene expression by qPCR. Bars represent fold increase over untreated controls. (N = 2, ± SD). (B) Reovirus up regulates nuclear translocation of NF-kB in BrCa cells. BrCa cells were incubated with 40 MOI of reovirus for 12 hours and nuclear and cyto solic extracts were prepared. NV or LV treated proteins were subjected to SDS/PAGE and blotted with anti NF-kBp65, actin and histone antibody.</p

PUMA and NF-kB Are Cell Signaling Predictors of Reovirus Oncolysis of Breast Cancer

<div><p>Background and purpose</p><p>Reovirus is a ubiquitous RNA virus that exploits aberrant signaling pathways for its replication. The oncolytic potential of reovirus against numerous cancers under pre-clinical/clinical conditions has been documented by us and others. Despite its proven clinical activity, the underlying mechanisms of reovirus oncolysis is still not well elucidated. If reovirus therapy is to be optimized for cancer, including breast cancer patients, it is imperative to understand the mechanisms of reovirus oncolysis, especially in treatment of resistant tumour.</p><p>Experimental approach and results</p><p>In the present study global gene expression profiling was utilized as a preliminary roadmap to tease-out pivotal molecules involved in reovirus induced apoptosis in breast cancer. Reovirus treated HTB133 and MCF7 breast cancer cells revealed transcriptional alteration of a defined subset of apoptotic genes and members of the nuclear factor-kappa B (NF-kB) family and p53 upregulated modulator of apoptosis (PUMA) were prominent. Since NF-kB can paradoxically suppress or promote apoptosis in cancer, the significance of NF-kB in reovirus oncolysis of breast cancer was investigated. Real time PCR analysis indicated a 2.9–4.3 fold increase in NF-kB p65 message levels following reovirus infection of MCF7 and HTB133, respectively. Nuclear translocation of NF-kB p65 protein was also dramatically augmented post reovirus treatment and correlated with enhanced DNA binding. Pharmacologic inhibition of NF-kB lead to oncolytic protection and significant down regulation of PUMA message levels. PUMA down regulation using siRNA suppressed reovirus oncolysis via significantly repressed apoptosis in p53 mutant HTB133 cells.</p><p>Conclusions</p><p>This study demonstrates for the first time that a prominent pathway of reovirus oncolysis of breast cancer is mediated through NF-kB and that PUMA upregulation is dependent on NF-kB activation. These findings represent potential therapeutic indicators of reovirus treatment in future clinical trials.</p></div

Transcriptional upregulation of apoptotic genes in HTB 133 and MCF breast cancer cells post reovirus infection^*.

<p>Transcriptional upregulation of apoptotic genes in HTB 133 and MCF breast cancer cells post reovirus infection^{<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0168233#t001fn001" target="_blank">*</a>}.</p

Reovirus induces apoptosis in BrCa cell lines.

<p>HTB133 and MCF7 cells were harvested at indicated time points following treatment with no virus (NV), dead virus (DV), or live virus (LV). Apoptosis was measured using flow cytometry via Annexin V binding, DNA fragmentation and Apo 2.7 expression. (N = 3, ± SD).</p

Molecular inhibition of PUMA leads to oncolytic protection.

<p><b>(A) PUMA down regulation via siRNA leads to suppression of PUMA protein expression.</b> HTB 133 cells were transfected with siRNA sequences against PUMA (Ambion) and total protein extracted was subjected to western analysis against PUMA and actin antibodies. <b>(B) Oncolytic protection by PUMA down regulation.</b> HTB 133 and MCF7 cells were transfected with siRNA sequences #2 against PUMA and infected with live or dead reovirus. Cells death was assayed via trypan blue (N = 3, ± SD).</p

Pharmacologic inhibition of NF-kB down regulates reovirus-induced PUMA gene expression in BrCa cells.

<p>Real time PCR was performed on RNA isolated from MCF7, HTB133 and HTB30 cells incubated with either media alone, CAPE (20μM), ALLN (10μM) for 3 hours before incubation with 40 MOI reovirus for 12 hours. Bars represent fold increase over untreated control cultures (N = 3, ± SD).</p

Functional aptitude of reovirus activated NK-kB in BrCa.

<p>(A) Reovirus induces NF-kB DNA binding in BrCa cells. BrCa cells were treated with either NV or LV for 12 hours or TNF (20 ng/ml) and nuclear extracts were prepared. 5 micro grams of nuclear proteins was incubated with an IR-dye labeled oligonucleotide consisting of the NF-kB consensus binding sequence and resolved by acrylamide gel electrophoresis and scanned in a LI-COR gel scanner. (B) NF-kB super shift complex is made up of p50 and p65. Nuclear proteins were incubated with 2 μl of super shift antibodies directed against NF-kB p50 or p65 before incubation with the NF-kB p65 probe. The mixtures were resolved in acrylamide gels and scanned as described under Fig 4A.</p

Reovirus infection of BrCa leads to upregulation of PUMA.

<p>(A) PUMA mRNA expression is induced in reovirus infected BrCa cell lines. MCF7 and HTB 133 cells were infected with 40 MOI of reovirus for 12, 24 and 48 hours. PUMA gene expression was quantified by real-time PCR. Bars represent fold increase over untreated control cultures (N = 3, ± SD). (B) Reovirus treatment up regulates PUMA protein expression in BrCa cells. BrCa cell lines were incubated with 40 MOI of reovirus for 12 hours and cytosolic extracts were prepared. 50 μg of virus treated (LV) or untreated (NV) proteins were subjected to SDS/PAGE and blotted with anti PUMA antibody and HRP linked secondary antibody.</p

Caspase 3/7 is activated during reovirus induced apoptosis of BrCa cells.

<p>BrCa cells were infected with No virus (NV), LV and DV at a MOI of 40 PFU/cell, or untreated (NV). Cells were harvested at 72h and <i>in situ</i> active caspase 3/7 activity was measured using flow cytometry.</p