Pyruvate anaplerosis is a mechanism of resistance to pharmacological glutaminase inhibition in triple-receptor negative breast cancer.

BACKGROUND: Glutamine serves as an important nutrient with many cancer types displaying glutamine dependence. Following cellular uptake glutamine is converted to glutamate in a reaction catalysed by mitochondrial glutaminase. This glutamate has many uses, including acting as an anaplerotic substrate (via alpha-ketoglutarate) to replenish TCA cycle intermediates. CB-839 is a potent, selective, orally bioavailable inhibitor of glutaminase that has activity in Triple receptor-Negative Breast Cancer (TNBC) cell lines and evidence of efficacy in advanced TNBC patients. METHODS: A panel of eleven breast cancer cell lines was used to investigate the anti-proliferative effects of the glutaminase inhibitors CB-839 and BPTES in different types of culture medium, with or without additional pyruvate supplementation. The abundance of the TCA cycle intermediate fumarate was quantified as a measure if TCA cycle anaplerosis. Pyruvate secretion by TNBC cultures was then assessed with or without AZD3965, a monocarboxylate transporter 1 (MCT1) inhibitor. Finally, two dimensional (2D) monolayer and three dimensional (3D) spheroid assays were used to compare the effect of microenvironmental growth conditions on CB-839 activity. RESULTS: The anti-proliferative activity of CB-839 in a panel of breast cancer cell lines was similar to published reports, but with a major caveat; growth inhibition by CB-839 was strongly attenuated in culture medium containing pyruvate. This pyruvate-dependent attenuation was also observed with a related glutaminase inhibitor, BPTES. Studies demonstrated that exogenous pyruvate acted as an anaplerotic substrate preventing the decrease of fumarate in CB-839-treated conditions. Furthermore, endogenously produced pyruvate secreted by TNBC cell lines was able to act in a paracrine manner to significantly decrease the sensitivity of recipient cells to glutaminase inhibition. Suppression of pyruvate secretion using the MCT1 inhibitor AZD3965, antagonised this paracrine effect and increased CB-839 activity. Finally, CB-839 activity was significantly compromised in 3D compared with 2D TNBC culture models, suggesting that 3D microenvironmental features impair glutaminase inhibitor responsiveness. CONCLUSION: This study highlights the potential influence that both circulating and tumour-derived pyruvate can have on glutaminase inhibitor efficacy. Furthermore, it highlights the benefits of 3D spheroid cultures to model the features of the tumour microenvironment and improve the in vitro investigation of cancer metabolism-targeted therapeutics.

Anticancer Characteristics of Fomitopsis pinicola Extract in a Xenograft Mouse Model-a Preliminary Study.

Introduction: Medicinal mushrooms have been used for the treatment of diseases and general promotion of health for many centuries. Recent pharmacological research into medicinal mushrooms has identified various therapeutic properties, with applications in modern medicine.Aim: To evaluate the anti-cancer activities of Fomitopsis pinicola (F. pinicola) alcoholic extract in an in vivo setting.Methods: The anti-tumour effect of the F. pinicola extract was tested in a xenograft immune-compromised Rag-1 mouse model. This was followed by RT-PCR and metabolomics analyses.Results: There were no observable differences in tumor growth between treated and non-treated groups. The bioactive components were not detected in the mouse plasma or the tumor site.Conclusions: The extract was poorly absorbed; this is likely due to the timing of treatment, dosage levels and modifications made to the extract where the alcohol-based solvent was replaced with water. This, in combination with fractionation studies which identified most anti-cancer compounds to be hydrophobic, largely explained the lack of anti-cancer activities in vivo.

Human Intervention Study to Assess the Effects of Supplementation with Olive Leaf Extract on Peripheral Blood Mononuclear Cell Gene Expression.

Olive leaf extract (OLE) has been used for many years for its putative health benefits, but, to date, scientific evidence for the basis of these effects has been weak. Although recent literature has described a link between ailments such as cardiovascular disease, diabetes and cancer and a protective effect of polyphenols in the OLE, the mode of action is still unclear. Here, we describe a double-blinded placebo (PBO)-controlled trial, in which gene expression profiles of peripheral blood mononuclear cells from healthy male volunteers (n = 29) were analysed to identify genes that responded to OLE, following an eight-week intervention with 20 mL daily consumption of either OLE or PBO. Differences between groups were determined using an adjusted linear model. Subsequent analyses indicated downregulation of genes important in inflammatory pathways, lipid metabolism and cancer as a result of OLE consumption. Gene expression was verified by real-time PCR for three genes (EGR1, COX-2 and ID3). The results presented here suggest that OLE consumption may result in health benefits through influencing the expression of genes in inflammatory and metabolic pathways. Future studies with a larger study group, including male and female participants, looking into direct effects of OLE on lipid metabolism and inflammation are warranted.

Identification of Potential Anticancer Activities of Novel Ganoderma lucidum Extracts Using Gene Expression and Pathway Network Analysis.

Ganoderma lucidum (lingzhi) has been used for the general promotion of health in Asia for many centuries. The common method of consumption is to boil lingzhi in water and then drink the liquid. In this study, we examined the potential anticancer activities of G. lucidum submerged in two commonly consumed forms of alcohol in East Asia: malt whiskey and rice wine. The anticancer effect of G. lucidum, using whiskey and rice wine-based extraction methods, has not been previously reported. The growth inhibition of G. lucidum whiskey and rice wine extracts on the prostate cancer cell lines, PC3 and DU145, was determined. Using Affymetrix gene expression assays, several biologically active pathways associated with the anticancer activities of G. lucidum extracts were identified. Using gene expression analysis (real-time polymerase chain reaction [RT-PCR]) and protein analysis (Western blotting), we confirmed the expression of key genes and their associated proteins that were initially identified with Affymetrix gene expression analysis.

Quinazolines as novel anti-inflammatory histone deacetylase inhibitors.

Histone deacetylase (HDAC) inhibitors regulate many biological responses, including anti-inflammatory and anti-cancer effects. We sought to identify novel classes of HDAC inhibitors from in-house compound libraries. Initially, compounds from 26 different structural classes that showed anti-inflammatory effects in a pre-screen in HEK293T cells were tested in vitro for HDAC inhibition, using a commercial fluorescence assay. The known HDAC inhibitors suberoylanilide hydroxamic acid (SAHA) and trichostatin A (TSA) were used as positive controls. Examples of three different structural classes (anilinoacridines, phenylpyrrolocarbazoles and benzofurylquinazolines) showed significant inhibition in the HDAC assay, and small subsets of these were also evaluated, seeking initial structure-activity relationships (SAR) for each class. Several of the most effective compounds from this HDAC screen were evaluated for their effects on the expression of the pro-inflammatory gene, IL1-alpha, and the cancer-related genes, p53, p21, E-cadherin and C-MYC. While the benzofurylquinazolines increased the expression level of the pro-inflammatory gene IL1-alpha as well as p21 and p53 in the PC3 cell line, a phenylpyrrolocarbazole had the converse effect on p53 expression. Several of the compounds showed in vitro HDAC inhibition ability in PC3, HCT116 and NIH-3T3 cell lines comparable to that of SAHA.