These changes were sufficient to inhibit tumour growth and showed that the combined treatment produced a substantially greater anti-tumour effect than either modality when used alone. Materials and Methods Cells DMS114, and H526 small cell lung cancer cells and HGC27 gastric cancer cells were maintained in RPMI1640 with 10% FCS and 1% L-glutamine (complete media). the breast  ovary  and prostate . In this model, tumour cells are able to induce oxidative stress, glycolysis, up-regulation Rabbit Polyclonal to Sumo1 of MCT4 and lactate efflux in cancer-associated fibroblasts, providing metabolic substrates for aerobic MCT1-expressing epithelial tumour cells. The transport of lactate is therefore critical to the maintenance of the symbiotic micro-environment and MCT1 is identified as the major transporter involved in lactate influx into tumour cells. In addition to the important functional role of MCT1 in tumour metabolism, MCT1 protein expression in tumours has been linked with variables associated with disease progression and prognosis in a variety of tumour types including breast  ovarian , gastric  and colorectal cancer . For these reasons, MCT1 is an attractive therapeutic target for inhibiting the metabolic interplay between cell populations within tumours. The non-specific MCT1 inhibitor -cyano-4-hydroxycinnamate (CHC) has been reported to produce anti-tumour effects by interfering with this metabolic coupling, by inhibiting lactate uptake into oxygenated tumour cells, increasing glucose SJ572403 uptake, and indirectly starving hypoxic tumour cells of glucose . In addition, CHC has been shown to increase necrosis and alter tumour volume [31-33]. However, CHC is not a specific MCT1 inhibitor and as a consequence these data should be interpreted with caution [34, 35]. Specific inhibitors with greater inhibitory potency and selectivity towards MCT1 have been developed for use in immunosuppression [36, 37], and these have been shown to influence lactate transport . Recent improvements on these compounds have resulted in the generation, by AstraZeneca, of AZD3965. This compound is a selective MCT1 inhibitor; it inhibits MCT1 with a binding affinity 1.6 nM and it is 6 fold selective over MCT2 and does not inhibit MCT3 or 4 at 10 M concentrations . In addition, the compound has good oral bioavailability, and it has entered phase I clinical trial for treatment of advanced solid tumours . In this study, we evaluate the metabolic and therapeutic effects of AZD3965 in small cell lung cancer and gastric cancer cell lines. In particular, we demonstrate the ability of AZD3965 to inhibit both lactate efflux and influx into cells and cause an increase in glycolysis and an up-regulation of glycolytic enzymes. These changes were sufficient to inhibit tumour growth and showed that the combined treatment produced a substantially greater anti-tumour effect than either modality when used alone. Materials and Methods Cells DMS114, and H526 small cell lung cancer cells and HGC27 gastric cancer cells were maintained in RPMI1640 with 10% FCS and 1% L-glutamine (complete media). Cell lines were chosen on the basis of relatively high sensitivity to AZD3965 . DMS114 and HDC27 SJ572403 cells were obtained from AstraZeneca and H526 cells were provided by the CR-UK Manchester Institute. All cells were subsequently authenticated while used in the University of Manchester laboratories by the use of an in-house DNA sequencing and authentication service. Cells were also shown to be mycoplasma free during the course of this work. For all experiments, cells were plated overnight prior to treatment with AZD3965 and/or different oxygen concentrations for a further 24hr (unless an alternative treatment time is indicated). Some experiments also included exposure to cobalt chloride as a hypoxia-mimetic. Cobalt chloride is able to mimic hypoxia by preventing HIF degradation, this is at least partially due to its occupation of the VHL-binding domain of HIF- 1 thus preventing its degradation . Glucose and Lactate uptake assay After treatment cells were washed prior to the addition of uptake cocktail consisting of buffer (25mM glucose, 137mM NaCl, 5.37mM KCl, 0.3mM Na2HPO4, 0.44mM KH2PO4, 4.17mM NaHCO3, 1.26mM CaCl2, 0.8mM MgSO4, 10mM HEPES SJ572403 pH 7.4) containing 100nM AZD3965 or equivalent volume of DMSO vehicle. For the glucose and lactate assays, 2mM glucose and 3H-2-deoxyglucose at an activity of 2Ci and 14C-lactate at an activity of 0. 2Ci were added respectively and incubated for one hour at 37C. Subsequently, the cocktail was removed and the cells washed and lysed in 0.5ml of lysis buffer (0.1% SDS and 0.1% Triton-X in HEPES buffer); 0.4ml of this was transferred to a scintillation vial with 4ml of scintillation fluid (Ecoscint A,.
These changes were sufficient to inhibit tumour growth and showed that the combined treatment produced a substantially greater anti-tumour effect than either modality when used alone