Combining the pan-aurora kinase inhibitor AMG 900 with histone deacetylase inhibitors enhances antitumor activity in prostate cancer.

Histone deacetylase inhibitors (HDACIs) are being tested in clinical trials for the treatment of solid tumors. While most studies have focused on the reexpression of silenced tumor suppressor genes, a number of genes/pathways are downregulated by HDACIs. This provides opportunities for combination therapy: agents that further disable these pathways through inhibition of residual gene function are speculated to enhance cell death in combination with HDACIs. A previous study from our group indicated that mitotic checkpoint kinases such as PLK1 and Aurora A are downregulated by HDACIs. We used in vitro and in vivo xenograft models of prostate cancer (PCA) to test whether combination of HDACIs with the pan-aurora kinase inhibitor AMG 900 can synergistically or additively kill PCA cells. AMG 900 and HDACIs synergistically decreased cell proliferation activity and clonogenic survival in DU-145, LNCaP, and PC3 PCA cell lines compared to single-agent treatment. Cellular senescence, polyploidy, and apoptosis was significantly increased in all cell lines after combination treatment. In vivo xenograft studies indicated decreased tumor growth and decreased aurora B kinase activity in mice treated with low-dose AMG 900 and vorinostat compared to either agent alone. Pharmacodynamics was assessed by scoring for phosphorylated histone H3 through immunofluorescence. Our results indicate that combination treatment with low doses of AMG 900 and HDACIs could be a promising therapy for future clinical trials against PCA.

[Shock in pregnancy: foetal distress may be the first symptom]. / Shock in de zwangerschap: foetale nood kan het eerste teken zijn.

Shock may be difficult to recognize in pregnant women due to the physiological changes that take place in the cardiovascular system. The first symptom of shock may be foetal distress. We present two patients to illustrate this condition. The first patient had an uncomplicated pregnancy until she awoke from a 'pop' in her abdomen followed by an acute feeling of illness. She was hemodynamically stable but because the foetal heart rate pattern was abnormal, an emergency caesarean section was performed. This revealed an intraperitoneal bleeding of the uterine artery in the right broad ligament, caused by ectopic decidualization. The second patient had severe symptomatic renal dilatation in pregnancy which was managed through percutaneous nephrostomy. Following the procedure she became hypotensive, tachycardic and hyperthermic, indications of septic shock. A neonate with signs of asphyxia was born by emergency caesarean section undertaken for acute foetal distress evident from the foetal heart rate pattern.

Analysis of the genomic response of human prostate cancer cells to histone deacetylase inhibitors.

Histone deacetylases (HDACs) have emerged as important targets for cancer treatment. HDAC-inhibitors (HDACis) are well tolerated in patients and have been approved for the treatment of patients with cutaneous T-cell lymphoma (CTCL). To improve the clinical benefit of HDACis in solid tumors, combination strategies with HDACis could be employed. In this study, we applied Analysis of Functional Annotation (AFA) to provide a comprehensive list of genes and pathways affected upon HDACi-treatment in prostate cancer cells. This approach provides an unbiased and objective approach to high throughput data mining. By performing AFA on gene expression data from prostate cancer cell lines DU-145 (an HDACi-sensitive cell line) and PC3 (a relatively HDACi-resistant cell line) treated with HDACis valproic acid or vorinostat, we identified biological processes that are affected by HDACis and are therefore potential treatment targets for combination therapy. Our analysis revealed that HDAC-inhibition resulted among others in upregulation of major histocompatibility complex (MHC) genes and deregulation of the mitotic spindle checkpoint by downregulation of genes involved in mitosis. These findings were confirmed by AFA on publicly available data sets from HDACi-treated prostate cancer cells. In total, we analyzed 375 microarrays with HDACi treated and non-treated (control) prostate cancer cells. All results from this extensive analysis are provided as an online research source (available at the journal's website and at http://luigimarchionni.org/HDACIs.html). By publishing this data, we aim to enhance our understanding of the cellular changes after HDAC-inhibition, and to identify novel potential combination strategies with HDACis for the treatment of prostate cancer patients.

Targeting prostate cancer cell lines with polo-like kinase 1 inhibitors as a single agent and in combination with histone deacetylase inhibitors.

Combinations of anticancer therapies with high efficacy and low toxicities are highly sought after. Therefore, we studied the effect of polo-like kinase 1 (Plk1) inhibitors on prostate cancer cells as a single agent and in combination with histone deacetylase (HDAC) inhibitors valproic acid and vorinostat. IC50s of Plk1 inhibitors BI 2536 and BI 6727 were determined in prostate cancer cells by MTS assays. Morphological and molecular changes were assessed by immunoblotting, immunofluorescence, flow cytometry, real-time RT-PCR, and pulldown assays. Efficacy of combination therapy was assessed by MTS and clonogenic assays. IC50 values in DU145, LNCaP, and PC3 cells were 50, 75, and 175 nM, respectively, for BI 2536 and 2.5, 5, and 600 nM, respectively, for BI 6727. Human prostate fibroblasts and normal prostate epithelial cells were unaffected at these concentrations. While DU145 and LNCaP cells were solely arrested in mitosis on treatment, PC3 cells accumulated in G2 phase and mitosis, suggesting a weak spindle assembly checkpoint. Combining Plk1 inhibitors with HDAC inhibitors had synergistic antitumor effects in vitro. DMSO-treated prostate cancer cells were used as controls to study the effect of Plk1 and HDAC inhibition. Plk1 inhibitors decreased proliferation and clonogenic potential of prostate cancer cells. Hence, Plk1 may serve as an important molecular target for inhibiting prostate cancer. Combining HDAC inhibitors with BI 2536 or BI 6727 may be an effective treatment strategy against prostate cancer.

[Anaphylaxis after iron dextran administration in a pregnant woman]. / Anafylaxie na ijzerdextraan bij een zwangere vrouw.

BACKGROUND: Iron deficiency is a frequent cause of anaemia in pregnancy and often results in fatigue and malaise. To prevent complications during labour, timely iron suppletion is important. CASE DESCRIPTION: A 30-year-old multiparous female presented at the outpatient clinic in her 38th week of this pregnancy because of fatigue and lightheadedness. She had been prescribed oral iron suppletion a month earlier but had not taken the tablets. Because her haemoglobin level had decreased to 6.3 mmol/l, it was decided to start her on intravenous iron dextran treatment. During administration of the test dose, the patient experienced acute dyspnoea and severe abdominal and back pain. Foetal bradycardia was observed and the patient underwent an emergency caesarean section. She delivered a healthy boy whose arterial pH was 7.05 (base excess: -7.6 mmol/l) and venous pH was 7.18 (base excess: -6.8 mmol/l). CONCLUSION: This case demonstrates that dextran anaphylaxis can occur, with potentially lethal consequences, even when no known underlying risk factors are present.

Downregulation of homologous recombination DNA repair genes by HDAC inhibition in prostate cancer is mediated through the E2F1 transcription factor.

BACKGROUND: Histone deacetylase inhibitors (HDACis) re-express silenced tumor suppressor genes and are currently undergoing clinical trials. Although HDACis have been known to induce gene expression, an equal number of genes are downregulated upon HDAC inhibition. The mechanism behind this downregulation remains unclear. Here we provide evidence that several DNA repair genes are downregulated by HDAC inhibition and provide a mechanism involving the E2F1 transcription factor in the process. METHODOLOGY/PRINCIPAL FINDINGS: Applying Analysis of Functional Annotation (AFA) on microarray data of prostate cancer cells treated with HDACis, we found a number of genes of the DNA damage response and repair pathways are downregulated by HDACis. AFA revealed enrichment of homologous recombination (HR) DNA repair genes of the BRCA1 pathway, as well as genes regulated by the E2F1 transcription factor. Prostate cancer cells demonstrated a decreased DNA repair capacity and an increased sensitization to chemical- and radio-DNA damaging agents upon HDAC inhibition. Recruitment of key HR repair proteins to the site of DNA damage, as well as HR repair capacity was compromised upon HDACi treatment. Based on our AFA data, we hypothesized that the E2F transcription factors may play a role in the downregulation of key repair genes upon HDAC inhibition in prostate cancer cells. ChIP analysis and luciferase assays reveal that the downregulation of key repair genes is mediated through decreased recruitment of the E2F1 transcription factor and not through active repression by repressive E2Fs. CONCLUSIONS/SIGNIFICANCE: Our study indicates that several genes in the DNA repair pathway are affected upon HDAC inhibition. Downregulation of the repair genes is on account of a decrease in amount and promoter recruitment of the E2F1 transcription factor. Since HDAC inhibition affects several pathways that could potentially have an impact on DNA repair, compromised DNA repair upon HDAC inhibition could also be attributed to several other pathways besides the ones investigated in this study. However, our study does provide insights into the mechanism that governs downregulation of HR DNA repair genes upon HDAC inhibition, which can lead to rationale usage of HDACis in the clinics.

Valproic acid causes dose- and time-dependent changes in nuclear structure in prostate cancer cells in vitro and in vivo.

Histone deacetylase inhibitors such as valproic acid (VPA) are promising anticancer agents that change the acetylation status of histones and loosen the chromatin structure. We assessed nuclear structure changes induced by VPA in prostate cancer LNCaP, CWR22R, DU145, and PC3 cell lines and xenografts and their potential use as a biomarker of treatment. In vitro tissue microarrays consisted of prostate cancer cell lines treated for 3, 7, or 14 days with 0, 0.6, or 1.2 mmol/L VPA. In vivo tissue microarrays consisted of cores from prostate cancer xenografts from nude mice treated for 30 days with 0.2% or 0.4% VPA in drinking water. Digital images of at least 200 Feulgen DNA-stained nuclei were captured using the Nikon CoolScope and nuclear alterations were measured. With a set of seven most frequently significant nuclear alterations (determined by univariate logistic regression analysis), control and VPA treatment nuclei were compared in vitro and in vivo. Depending on the cell line, area under the curve-receiver operating characteristics ranged between 0.6 and 0.9 and were dose- and time-dependent both in vitro and in vivo. Also, VPA treatment caused significant nuclear alterations in normal drug-filtering organs (liver and kidney tissue). In vitro and in vivo VPA treatment of prostate cancer cell lines results in significant dose- and time-dependent changes in nuclear structure. Further, VPA induces nuclear structural changes in normal liver and kidney tissue, which likely reflects a natural physiologic response. Therefore, nuclear structural alterations may serve as a biomarker for histone deacetylase inhibitor treatment.

Hybrids of aneuploid human cancer cells permit complementation of simple and complex cancer defects.

Causes for the complex phenotypes of cancers, such as altered differentiation, invasion and metastasis, are not known, and multigenic defects are likely. In contrast, well-defined deficiencies, such as those affecting DNA-repair mechanisms and enzymatic pathways, are simple, typically caused by one or a few gene mutations. Complementation by introducing defined genetic elements is used to study simple cancer phenotypes, while complementation by the fusion of whole cells is employed occasionally for complex ones. Hybrids formed solely from the common lines (aneuploid due to chromosomal instability, CIN) are rarely reported. We created stable hybrids of two CIN lines, producing a nearly complete genetic sum of the parental karyotypes. Complementation of a simple cancer phenotype, a Fanconi anemia pathway defective in both parental lines, occurred in all hybrids, restoring the normal drug-resistance phenotype. The grossly defective mitotic spindle checkpoint present in both parental lines was partially corrected in some hybrids, supporting a multigenic origin rather than a single gene defect. Using Affymetrix 100K SNP chips, we mapped chromosomal loci differing among the phenotypically distinct hybrid clones. Fusing CIN cell lines to form mapped hybrids offers new tools for positional cloning or classification of simple and complex cancer phenotypes, including mechanical defects and altered drug responses.

A multiple-loop, double-cube microarray design applied to prostate cancer cell lines with variable sensitivity to histone deacetylase inhibitors.

PURPOSE: Although microarray technology has been widely adopted by the scientific community, analysis of the ensuing data remains challenging. In this article, we present our experience with a complex design microarray experiment on resistance mechanisms of histone deacetylase inhibitors (HDACI). EXPERIMENTAL DESIGN: To improve our understanding of the underlying mechanism of HDACI resistance in prostate cancer cells, we designed a novel "multiple-loop, double-cube" cDNA microarray experiment. In the experiment of 22 arrays, DU145 and PC3 cells were treated with two different HDACIs (vorinostat and valproic acid) and incubation periods (48 and 96 h). Preprocessing included exploratory analyses of the quality of the arrays and intensity-dependent within-array Loess normalization. An ANOVA model was used for inference. The results were validated by Western blot analysis of known treatment targets. RESULTS: Treatment of PC3 and DU145 cells with HDACIs caused 2.8% to 10% (P<0.001) differential expression across conditions; 51% to 73% of these genes were up-regulated and 28% to 49% were down-regulated. The extent of differential expression was associated with cell line (DU145>PC3), HDACI (valproic acid >or= vorinostat), and duration of treatment (96>48 h). We identified known and new treatment targets involved in cell cycle and apoptosis. CONCLUSION: A multiple-loop, double-cube microarray design can be used to identify HDACI-induced changes in gene expression possibly related to drug resistance.

Multiple Molecular pathways explain the anti-proliferative effect of valproic acid on prostate cancer cells in vitro and in vivo.

BACKGROUND: Valproic acid (VPA), is a drug approved by the FDA for epilepsy and bipolar disorders. It is a known Histone Deacetylase Inhibitor (HDACI). We tested VPA, for its anti-proliferative activity in prostate cancer (PCa) cell lines in vitro and in vivo. METHODS: DU-145 and PC-3 PCa cell lines were cultured with different doses of VPA. Cells were examined for their viability, cell cycle status and expression of cell cycle arrest, and proliferation markers. Nude mice bearing xenografts of human PCa cell lines, DU-145, and PC-3, were administered VPA in their drinking water. RESULTS: VPA displayed a dose- and time-dependent anti-proliferative effect on DU-145 and PC-3 PCa cell lines in vitro. A sustained effect of the drug was seen on cell cycle arrest even at 24 hr after removal of the drug, after which the effects returned to the basal state. Administration of 0.4% w/v VPA in drinking water (resulting in 0.4 mM VPA, in plasma) was effective in inducing growth arrest, cell death, and senescence in vivo and was also anti-angiogenic. The activation of all or some of these anti-proliferative pathways may be contingent on acetylation status of histones, confirmed by detection of increased acetyl-H3K9 in VPA-treated samples when compared with untreated controls. Pharmacodynamic studies showed an increase in expression of p21 and decrease in PCNA in xenografts of VPA-treated mice compared with protein expression in untreated controls. CONCLUSIONS: VPA may be functioning as an HDACI to inhibit growth of PCa cells in vitro and in vivo by modulating multiple pathways including cell cycle arrest, apoptosis, angiogenesis, and senescence.