Non-Phosphorylatable PEA-15 Sensitises SKOV-3 Ovarian Cancer Cells to Cisplatin

The efficacy of cisplatin-based chemotherapy in ovarian cancer is often limited by the development of drug resistance. In most ovarian cancer cells, cisplatin activates extracellular signal-regulated kinase1/2 (ERK1/2) signalling. Phosphoprotein enriched in astrocytes (PEA-15) is a ubiquitously expressed protein, capable of sequestering ERK1/2 in the cytoplasm and inhibiting cell proliferation. This and other functions of PEA-15 are regulated by its phosphorylation status. In this study, the relevance of PEA-15 phosphorylation state for cisplatin sensitivity of ovarian carcinoma cells was examined. The results of MTT-assays indicated that overexpression of PEA-15AA (a non-phosphorylatable variant) sensitised SKOV-3 cells to cisplatin. Phosphomimetic PEA-15DD did not affect cell sensitivity to the drug. While PEA-15DD facilitates nuclear translocation of activated ERK1/2, PEA-15AA acts to sequester the kinase in the cytoplasm as shown by Western blot. Microarray data indicated deregulation of thirteen genes in PEA-15AA-transfected cells compared to non-transfected or PEA-15DD-transfected variants. Data derived from The Cancer Genome Atlas (TCGA) showed that the expression of seven of these genes including EGR1 (early growth response protein 1) and FLNA (filamin A) significantly correlated with the therapy outcome in cisplatin-treated cancer patients. Further analysis indicated the relevance of nuclear factor erythroid 2-related factor 2/antioxidant response element (Nrf2/ARE) signalling for the favourable effect of PEA-15AA on cisplatin sensitivity. The results warrant further evaluation of the PEA-15 phosphorylation status as a potential candidate biomarker of response to cisplatin-based chemotherapy. View Full-Tex

Mechanism of anti-tumor activity of adenovirus type 5 E1A in ovarian cancer

The adenovirus type 5 E1A gene was originally developed as a gene therapy to inhibit tumorigenicity of HER-2-overexpressing cells by transcriptional downregulation of HER-2. Our goal is to improve the overall efficacy of E1A gene therapy. To achieve this goal, we have conducted two preclinical experiments. First, we hypothesized that Bcl-2 overexpressing ovarian cancer is resistant to E1A gene therapy. This hypothesis is based on that the 19 kDa protein product of the adenoviral E1B gene which is homologous to Bcl-2 inhibits E1A-induced apoptosis. Treating high Bcl-2-xpressing cells with E1A in combination with an antisense oligonucleotide to Bcl-2 (Bcl-2-ASO) resulted in a significant decrease in cell viability due to an increased rate of apoptosis relative to cells treated with E1A alone. In an ovarian cancer xenograft model, mice implanted with low HER-2, high Bcl-2 cells, treated with E1A plus Bcl-2-ASO led to prolonged survival. Bcl-2 thus may serve as a predictive molecular marker enabling us to select patients with ovarian cancer who will benefit significantly from E1A gene therapy. Second, we elucidated the molecular mechanism governing the anti-tumor effect of E1A in ovarian cancer to identify a more potent tumor suppressor gene. We identified PEA-15 (phospho-protein enriched in astrocytes) upregulated in E1A transfected low HER-2-expressing OVCAR-3 ovarian cancer cell, which showed decreased cell proliferation. PEA-15 moved ERK from the nucleus to the cytoplasm and inhibited ERK-dependent transcription and proliferation. Using small interfering RNA to knock down PEA-15 expression in OVCAR-3 cells made to constitutively express E1A resulted in accumulation of phosphoERK in the nucleus, an increase in Elk-1 activity, DNA synthesis, and anchorage-independent growth. PEA-15 also independently suppressed colony formation in some breast and ovarian cancer cell lines in which E1A is known to have anti-tumor activity. We conclude that the anti-tumor activity of E1A depends on PEA-15. In summary, (1) Bcl-2 may serve as a predictive molecular marker of E1A gene therapy, allowing us to select patients and improve efficacy of E1A gene therapy. (2) PEA-15 was identified as a component of the molecular mechanism governing the anti-tumor activity of E1A in ovarian cancer, (3) PEA-15 may be developed as a novel therapeutic gene

Celecoxib decreases the viability of MDA-MB-231-and MDA-MB-435-stable transfectants

<p><b>Copyright information:</b></p><p>Taken from "Adenovirus type 5 -induced apoptosis in COX-2-overexpressing breast cancer cells"</p><p>Breast cancer research : BCR 2007;9(4):R41-R41.</p><p>Published online 5 Jul 2007</p><p>PMCID:PMC2206712.</p><p></p> MTT assays after a 5-day exposure to 0–60 μM celecoxib indicate substantial reductions in cell viability in all variants of the MDA-MB-231 and MDA-MB-435 cell lines (stable transfectants, vector control, and parental control cells). Each point represents means from tests performed in quadruplicate; the bars are standard deviations. In both cell lines, the transfectants were more sensitive than the vector control or parental control cells. MTT assays in all variants of the SKOV3.ip1 and MCF-7 cell lines. The transfectants have no difference to sensitivity for celecoxib in both cell lines. Trypan blue assays after a 5-day exposure to 40 μM celecoxib show substantial reductions in viability of MDA-MB-231 and MDA-MB-435 cells. Values shown are normalized to the viability of the control (untreated) cells. Each bar represents means from tests performed in quadruplicate; bars are standard deviations. values are from two-tailed paired tests

Celecoxib downregulated COX-2 protein expression in all MDA-MB-231 and MDA-MB-435 variants, but celecoxib downregulated Bcl-2 expression in only the MDA-MB-435-stable transfectants

<p><b>Copyright information:</b></p><p>Taken from "Adenovirus type 5 -induced apoptosis in COX-2-overexpressing breast cancer cells"</p><p>Breast cancer research : BCR 2007;9(4):R41-R41.</p><p>Published online 5 Jul 2007</p><p>PMCID:PMC2206712.</p><p></p> () Western blots of MDA-MB-231 and MDA-MB-435 cells treated with 0 or 40 μM celecoxib for 5 days and tested for COX-2 and Bcl-2. Percentages indicate differences relative to the 0 μM control samples. Protein expression was considered to be downregulated if the treated condition was at least 20% less than the control (untreated) condition. () Time course of Bcl-2 expression after treatment with 0 or 40 μM celecoxib in MDA-MB-435-and MDA-MB-231-stable transfectants. Bcl-2 was suppressed at both 72 and 96 h in the MDA-MB-435-stable transfectants but was not suppressed in the MDA-MB-231-stable transfectants. () Transfection of MDA-MB-435 cells with Bcl-2 DNA (+) or a control DNA (-) led to overexpression of Bcl-2 in all variants. () MDA-MB-435-cells made to overexpress Bcl-2 and non-Bcl-2-overexpressing cells were treated with 0 or 40 μM celecoxib for 5 days, and cell viability was determined with a trypan-blue assay. Bcl-2 overexpression did not restore sensitivity to celecoxib (= 0.11)

A Novel Second-generation MELK specific inhibitor targets triple negative inflammatory breast cancer (TN-IBC)

https://openworks.mdanderson.org/sumexp21/1142/thumbnail.jp

Celecoxib enhances apoptosis of MDA-MB-231-and MDA-MB-435-stable transfectants

<p><b>Copyright information:</b></p><p>Taken from "Adenovirus type 5 -induced apoptosis in COX-2-overexpressing breast cancer cells"</p><p>Breast cancer research : BCR 2007;9(4):R41-R41.</p><p>Published online 5 Jul 2007</p><p>PMCID:PMC2206712.</p><p></p> Cell cycle distribution of MDA-MB-231-and MDA-MB-435-cells was detected by fluorescence-activated cell sorting after a 5-day exposure to 0 or 40 μM celecoxib. The percentage of cells in sub-G(apoptosis) appears at the upper right of each graph. Western blots of MDA-MB-231 and MDA-MB-435 cells treated with 0 or 40 μM celecoxib for 5 days and tested for cleaved caspase-9 (cl-cas-9), uncleaved and cleaved caspase-8 (cl-cas-8), PARP (uncleaved and cleaved), E1A, and actin. Cleaved PARP and cleaved caspase-9 levels were higher after celecoxib treatment in the MDA-MB-231-and MDA-MB-435-stable transfectants, but expression of cleaved caspase-8 (cl-cas-8) did not change

COX-2 protein expression in breast and ovarian cancer cell lines stably transfected with

<p><b>Copyright information:</b></p><p>Taken from "Adenovirus type 5 -induced apoptosis in COX-2-overexpressing breast cancer cells"</p><p>http://breast-cancer-research.com/content/9/4/R41</p><p>Breast cancer research : BCR 2007;9(4):R41-R41.</p><p>Published online 5 Jul 2007</p><p>PMCID:PMC2206712.</p><p></p> MDA-MB-231-stable transfectants produced the greatest amounts of COX-2; MDA-MB-435-cells produced 78% of that amount, but the SKOV3.ip1-and MCF-7-cells produced only 6% of that amount. MDA-MB-231-cells expressed slightly more E1A than the other three cell lines. COX-2 protein expression level between the stable transfectants and their corresponding vector control cells or parent cells. If the COX-2 expression levels of each transfectant is defined as 100%, the corresponding COX-2 expression levels of the vector controls were as follows: 65% for MDA-MB-231, 144% for MDA-MB-435, 71% for SKOV3.ip1 and 67% for MCF-7

Expression of histone deacetylase (HDAC) family members in bortezomib-refractory multiple myeloma and modulation by panobinostat

Aim: Multiple myeloma (MM) is a hematological malignancy of antibody-producing mature B cells or plasma cells. The proteasome inhibitor, bortezomib, was the first-in-class compound to be FDA approved for MM and is frequently utilized in induction therapy. However, bortezomib refractory disease is a major clinical concern, and the efficacy of the pan-histone deacetylase inhibitor (HDACi), panobinostat, in bortezomib refractory disease indicates that HDAC targeting is a viable strategy. Here, we utilized isogenic bortezomib resistant models to profile HDAC expression and define baseline and HDACi-induced expression patterns of individual HDAC family members in sensitive vs. resistant cells to better understanding the potential for targeting these enzymes.Methods: Gene expression of HDAC family members in two sets of isogenic bortezomib sensitive or resistant myeloma cell lines was examined. These cell lines were subsequently treated with HDAC inhibitors: panobinostat or vorinostat, and HDAC expression was evaluated. CRISPR/Cas9 knockdown and pharmacological inhibition of specific HDAC family members were conducted.Results: Interestingly, HDAC6 and HDAC7 were significantly upregulated and downregulated, respectively, in bortezomib-resistant cells. Panobinostat was effective at inducing cell death in these lines and modulated HDAC expression in cell lines and patient samples. Knockdown of HDAC7 inhibited cell growth while pharmacologically inhibiting HDAC6 augmented cell death by panobinostat.Conclusion: Our data revealed heterogeneous expression of individual HDACs in bortezomib sensitive vs. resistant isogenic cell lines and patient samples treated with panobinostat. Cumulatively our findings highlight distinct roles for HDAC6 and HDAC7 in regulating cell death in the context of bortezomib resistance