Pyrrolo-1,5-benzoxazepines:a new class of apoptotic agents

Some members of a series of novel pyrrolo-1,5-benzoxazepines (PBOXs) potently induce apoptosis in a number of human cancerous cell lines including HL-60 cells and the drug-resistant chronic myelogenous leukaemia cell line, K562. The apoptotic induction seems to be independent of the mitochondrial peripheral-type benzodiazepine receptor (PBR), which binds these PBOXs with high affinity, due to a lack of correlation between their affinities for the receptor and their apoptotic potencies and their high apoptotic activity in PBR-deficient cells. PBOX-6, a potent member of the series, induces a transient activation of c-Jun N-terminal kinase (JNK) in a dose-dependent manner, which correlates with induction of apoptosis. Expression of a cytoplasmic inhibitor of the JNK signal transduction pathway, Jip-1, prevents JNK activity and significantly reduces the extent of apoptosis induced by PBOX-6. This demonstrates the requirement for JNK in the cellular response to this apoptotic agent. In addition, PBOX-6 activates caspase-3-like proteases in K562 and HL-60 cells. The caspase-3 inhibitor, Z-Asp-Glu-Val-Asp-fluoromethylketone (z-DEVD-fmk), blocks caspase-3-like protease activity in both cell types but only prevents PBOX-6-induced apoptosis in HL-60 cells, suggesting that the requirement for caspase-3-like proteases in the apoptotic pathway is dependent on the cell type.

ER stress-mediated autophagy promotes Myc-dependent transformation and tumor growth

The proto-oncogene c-Myc paradoxically activates both proliferation and apoptosis. In the pathogenic state, c-Myc-induced apoptosis is bypassed via a critical, yet poorly understood escape mechanism that promotes cellular transformation and tumorigenesis. The accumulation of unfolded proteins in the ER initiates a cellular stress program termed the unfolded protein response (UPR) to support cell survival. Analysis of spontaneous mouse and human lymphomas demonstrated significantly higher levels of UPR activation compared with normal tissues. Using multiple genetic models, we demonstrated that c-Myc and N-Myc activated the PERK/eIF2α/ATF4 arm of the UPR, leading to increased cell survival via the induction of cytoprotective autophagy. Inhibition of PERK significantly reduced Myc-induced autophagy, colony formation, and tumor formation. Moreover, pharmacologic or genetic inhibition of autophagy resulted in increased Myc-dependent apoptosis. Mechanistically, we demonstrated an important link between Myc-dependent increases in protein synthesis and UPR activation. Specifically, by employing a mouse minute (L24+/-) mutant, which resulted in wild-type levels of protein synthesis and attenuation of Myc-induced lymphomagenesis, we showed that Myc-induced UPR activation was reversed. Our findings establish a role for UPR as an enhancer of c-Myc-induced transformation and suggest that UPR inhibition may be particularly effective against malignancies characterized by c-Myc overexpression.

Survival or death: a dual role of autophagy in stress-induced pericyte loss in diabetic retinopathy

Aims/hypothesis
Intra-retinal extravasation and modification of LDL have been implicated in diabetic retinopathy: autophagy may mediate these effects.
Methods
Immunohistochemistry was used to detect autophagy marker LC3B in human and murine diabetic and non-diabetic retinas. Cultured human retinal capillary pericytes (HRCPs) were treated with in vitro-modified heavily-oxidised glycated LDL (HOG-LDL) vs native LDL (N-LDL) with or without autophagy modulators: green fluorescent protein–LC3 transfection; small interfering RNAs against Beclin-1, c-Jun NH(2)-terminal kinase (JNK) and C/EBP-homologous protein (CHOP); autophagy inhibitor 3-MA (5 mmol/l) and/or caspase inhibitor Z-VAD-fmk (100 μmol/l). Autophagy, cell viability, oxidative stress, endoplasmic reticulum stress, JNK activation, apoptosis and CHOP expression were assessed by western blots, CCK-8 assay and TUNEL assay. Finally, HOG-LDL vs N-LDL were injected intravitreally to STZ-induced diabetic vs control rats (yielding 50 and 200 mg protein/l intravitreal concentration) and, after 7 days, retinas were analysed for ER stress, autophagy and apoptosis.
Results
Intra-retinal autophagy (LC3B staining) was increased in diabetic vs non-diabetic humans and mice. In HRCPs, 50 mg/l HOG-LDL elicited autophagy without altering cell viability, and inhibition of autophagy decreased survival. At 100–200 mg/l, HOG-LDL caused significant cell death, and inhibition of either autophagy or apoptosis improved survival. Further, 25–200 mg/l HOG-LDL dose-dependently induced oxidative and ER stress. JNK activation was implicated in autophagy but not in apoptosis. In diabetic rat retina, 50 mg/l intravitreal HOG-LDL elicited autophagy and ER stress but not apoptosis; 200 mg/l elicited greater ER stress and apoptosis.
Conclusions
Autophagy has a dual role in diabetic retinopathy: under mild stress (50 mg/l HOG-LDL) it is protective; under more severe stress (200 mg/l HOG-LDL) it promotes cell death.

Insulin-like growth factor binding protein-3 mediates vascular repair by enhancing nitric oxide generation

Insulin-like growth factor binding protein (IGFBP)-3 modulates vascular development by regulating endothelial progenitor cell (EPC) behavior, specifically stimulating EPC cell migration. This study was undertaken to investigate the mechanism of IGFBP-3 effects on EPC function and how IGFBP-3 mediates cytoprotection following vascular injury.

The Absolute Configuration for Inthomycin C: Revision of Previously Published Work with a Reinstatement of the (3R)-Configuration for (-)-Inthomycin C

Abstract Image

Stereochemical evidence is presented to demonstrate that (−)-inthomycin C has (3R)- and not (3S)-stereochemistry. Careful reappraisal of the previously published work2−5 now indicates that the Hatakeyama, Hale, Ryu, and Taylor teams all have synthesized (−)-(3R)-inthomycin C. The newly measured [α]D of pure (−)-(3R)-inthomycin C (98% ee) is −7.9 (c 0.33, CHCl3) and not −41.5 (c 0.1, CHCl3) as was previously reported in 2012.

O-GlcNAc transferase integrates metabolic pathways to regulate the stability of c-MYC in human prostate cancer cells

Metabolic disruptions that occur widely in cancers offer an attractive focus for generalized treatment strategies. The hexosamine biosynthetic pathway (HBP) senses metabolic status and produces an essential substrate for O-linked β-N-acetylglucosamine transferase (OGT), which glycosylates and thereby modulates the function of its target proteins. Here, we report that the HBP is activated in prostate cancer cells and that OGT is a central regulator of c-Myc stability in this setting. HBP genes were overexpressed in human prostate cancers and androgen regulated in cultured human cancer cell lines. Immunohistochemical analysis of human specimens (n = 1987) established that OGT is upregulated at the protein level and that its expression correlates with high Gleason score, pT and pN stages, and biochemical recurrence. RNA interference-mediated siliencing or pharmacologic inhibition of OGT was sufficient to decrease prostate cancer cell growth. Microarray profiling showed that the principal effects of OGT inhibition in prostate cancer cells were related to cell-cycle progression and DNA replication. In particular, c-MYC was identified as a candidate upstream regulator of OGT target genes and OGT inhibition elicited a dose-dependent decrease in the levels of c-MYC protein but not c-MYC mRNA in cell lines. Supporting this relationship, expression of c-MYC and OGT was tightly correlated in human prostate cancer samples (n = 1306). Our findings identify HBP as a modulator of prostate cancer growth and c-MYC as a key target of OGT function in prostate cancer cells.

c-Met inhibition in a HOXA9/Meis1 model of CN-AML

BACKGROUND: Hematopoiesis is a paradigm for developmental processes, hierarchically organized, with stem cells at its origin. Hematopoietic stem cells (HSCs) replenish progenitor and precursor cells of multiple lineages, which normally differentiate into short-lived mature circulating cells. Hematopoiesis has provided insight into the molecular basis of tissue homeostasis and malignancy. Malignant hematopoiesis, in particular acute myeloid leukemia (AML), results from impaired development or differentiation of HSCs and progenitors. Co-overexpression of HOX and TALE genes, particularly the HOXA cluster and MEIS1, is associated with AML. Clinically relevant models of AML are required to advance drug development for an aging patient cohort.

RESULTS: Molecular analysis identified altered gene, microRNA, and protein expression in HOXA9/Meis1 leukemic bone marrow compared to normal controls. A candidate drug screen identified the c-Met inhibitor SU11274 for further analysis. Altered cell cycle status, apoptosis, differentiation, and impaired colony formation were shown for SU11274 in AML cell lines and primary leukemic bone marrow.

CONCLUSIONS: The clonal HOXA9/Meis1 AML model is amenable to drug screening analysis. The data presented indicate that human AML cells respond in a similar manner to the HOXA9/Meis1 cells, indicating pre-clinical relevance of the mouse model.

Prognostic role of the LCS6 KRAS variant in locally advanced rectal cancer: results of the EXPERT-C trial.

Background: Lethal-7 (let-7) is a tumour suppressor miRNA which acts by down-regulating several oncogenes including KRAS. A single-nucleotide polymorphism (rs61764370, T > G base substitution) in the let-7 complementary site 6 (LCS-6) of KRAS mRNA has been shown to predict prognosis in early-stage colorectal cancer (CRC) and benefit from anti-epidermal growth factor receptor monoclonal antibodies in metastatic CRC. Patients and methods: We analysed rs61764370 in EXPERT-C, a randomised phase II trial of neoadjuvant CAPOX followed by chemoradiotherapy, surgery and adjuvant CAPOX plus or minus cetuximab in locally advanced rectal cancer. DNA was isolated from formalin-fixed paraffin-embedded tumour tissue and genotyped using a PCR-based commercially available assay. Kaplan–Meier method and Cox regression analysis were used to calculate survival estimates and compare treatment arms. Results: A total of 155/164 (94.5%) patients were successfully analysed, of whom 123 (79.4%) and 32 (20.6%) had the LCS-6 TT and LCS-6 TG genotype, respectively. Carriers of the G allele were found to have a statistically significantly higher rate of complete response (CR) after neoadjuvant therapy (28.1% versus 10.6%; P = 0.020) and a trend for better 5-year progression-free survival (PFS) [77.4% versus 64.5%: hazard ratio (HR) 0.56; P = 0.152] and overall survival (OS) rates (80.3% versus 71.9%: HR 0.59; P = 0.234). Both CR and survival outcomes were independent of the use of cetuximab. The negative prognostic effect associated with KRAS mutation appeared to be stronger in patients with the LCS-6 TT genotype (HR PFS 1.70, P = 0.078; HR OS 1.79, P = 0.082) compared with those with the LCS-6 TG genotype (HR PFS 1.33, P = 0.713; HR OS 1.01, P = 0.995). Conclusion: This analysis suggests that rs61764370 may be a biomarker of response to neoadjuvant treatment and an indicator of favourable outcome in locally advanced rectal cancer possibly by mitigating the poor prognosis of KRAS mutation. In this setting, however, this polymorphism does not appear to predict cetuximab benefit.