[18F]2-Fluoro-2-deoxy-D-glucose incorporation by AGS gastric adenocarcinoma cells in vitro during response to epirubicin, cisplatin and 5-fluorouracil

Decreased tumour [18F]2-fluoro-2-deoxy-D-glucose (18FDG) incorporation is related to response however its significance at the cell level in gastro-oesophageal cancer and how it relates to cell death is unknown. Here human gastric adenocarcinoma (AGS) cells were treated with lethal dose 10 and 50 (LD10 and LD50), determined by using the MTT assay, of the three drugs, epirubicin, 5-fluorouracil and cisplatin, commonly used in the treatment of patients with gastro-oesophageal cancer. 18FDG incorporation was determined after 48 and 72 h of treatment with each drug and related to drug-induced changes in glucose transport, hexokinase activity, cell cycle distribution and annexin V-PE binding (a measure of apoptosis). Treatment of cells for 48 and 72 h with LD50 doses of cisplatin resulted in reductions in 18FDG incorporation of 27 and 25% respectively and of 5-fluorouracil reduced 18FDG incorporation by 34 and 33% respectively: epirubicin treatment reduced incorporation by 30 and 69% respectively. Cells that had been treated for 72 h with each drug were incubated in drug-free media for a further 6 days to determine their ability to recover. Comparison of the ability to recover from the chemotherapy agent, with 18FDG incorporation before the recovery period allowed an assessment of the predictive ability of 18FDG incorporation. Cells treated with either 5-fluorouracil or cisplatin demonstrated recovery on removal of the drug. In contrast, cells treated with epirubicin did not recover corresponding with the greatest 72 h treatment decrease in 18FDG incorporation. In contrast to adherent cells treated with cisplatin or 5-fluorouracil, adherent epirubicin-treated cells also exhibited very high levels of apoptosis. Glucose transport was decreased after each treatment whilst hexokinase activity was only decreased after 72 h of treatment with each drug. There was no consistent relationship observed between 18FDG incorporation and cell cycle distribution. Our results show that at the tumour cell level in gastric tumour cells, decreased 18FDG incorporation and glucose transport, accompanies therapeutic growth inhibition. 18FDG incorporation is particularly diminished in cells exhibiting apoptosis.

Combination chemotherapy may improve the length and quality of survival in a proportion of patients with advanced gastrooesophageal cancer. With currently available chemotherapy regimens, only 40 -60% of patients will respond to treatment (Gilbert et al, 2002). Present chemotherapy regimens for gastro-oesophageal cancer include epirubicin, cisplatin and 5-fluorouracil (5-FU). If patients are to avoid unnecessary treatment with particularly toxic agents, it is important that non-responders are identified at an early stage.
A number of studies (Couper et al, 1998;Weber et al, 2001;Kroep et al, 2003;Ott et al, 2003) have demonstrated that a reduction in tumour uptake of the glucose analogue, [ 18 F]2-fluoro-2-deoxy-D-glucose ( 18 FDG), is observed during and upon completion of chemotherapy in gastro-oesophageal cancer. Although decreased tumour 18 FDG uptake has been shown to be associated with response (and may in fact predict tumour response), its significance at the gastro-oesophageal tumour cell level is not known. Response to chemotherapy, as seen on 18 FDG-PET with a corresponding reduction in uptake in solid tumours, may be related to a decrease in tumour cellularity or a decrease in 18 FDG uptake per cell, or a combination of both. In solid tumours, tumour cells can recover and it is not known if this decreased 18 FDG uptake during response is due to modulation in 18 FDG uptake by cells that will not recover. This reduction in cellular uptake of 18 FDG may occur prior to reduction in tumour volume and therefore may reflect apoptosis. To further confuse matters, a flare phenomenon has been recorded in the early period following exposure to chemotherapy (Basu and Alavi, 2007). In addition chemotherapy may alter 18 FDG cellular uptake by hexokinase (HK) and/or glucose transport modulation.
The fluorinated glucose analogue, 18 FDG, is transported into tumour cells via a family of glucose transporter proteins, then phosphorylated by the enzyme HK to 18 FDG-6-phosphate after which it undergoes little further metabolism. Due to low levels of G-6-Pase in tumour cells this is considered an irreversible reaction (Weber and Cantero, 1955;Warburg, 1956;Gallagher et al, 1978).
We have determined the effect of three commonly used chemotherapeutic agents (epirubicin, cisplatin and 5-FU) in the treatment of upper gastro-intestinal tumours on the cellular incorporation of 18 FDG and on steps associated with its incorporation, that is, glucose transport and HK activity. As tumour cells within solid tumours are likely to be exposed to different drug concentrations, we have treated cells with both lethal dose 50 (LD 50 ) and drug doses that have a low cell growth inhibition (LD 10 , 5 -10% cell death). Previous work on AGS (human gastric adenocarcinoma) cells has shown that they can recover from treatment with cytotoxic doses of combinational regimes of 5-FU and cisplatin but not when epirubicin is included in the combination (Couper and Park, 2003). However, the design of Coupers' study meant that this effect could not be specifically related to epirubicin rather than triple combination chemotherapy. Our study evaluated the effect of each individual chemotherapeutic agent. Growth curves for AGS cells were established after exposure to each epirubicin, cisplatin and 5-FU, with LD 10 and LD 50 established for each agent at 48 h. Growth curves were taken to 216 h to determine the extent to which cells were irreversibly damaged by the chemotherapeutic agents. All subsequent experiments on 18 FDG uptake and its relationships to glucose transport, HK activity, cell viability, cell cycling and apoptosis were undertaken at 48 and 72 h.
The aims of this study were to elucidate the effect, on cellular (AGS cells) 18 FDG incorporation, of exposure to three commonly used chemotherapeutic agents in the treatment of gastrooesophageal cancer and to determine the mechanisms behind these changes in 18 FDG cellular incorporation and their relationship with apoptosis.
A seeding density of 7500 cells per 0.35 cm 2 , based on previous work (Couper and Park, 2003), was used for all experiments.
Cells were plated in HAM F-12 media, at optimal seeding density, and returned to the incubator for 24 h to allow cell adherence to the plate. Media supplemented with varying concentrations of the chemotherapeutic agents epirubicin (Pharmacia and Upjohn Ltd., Milton Keynes, UK) (0.0039 -0.5 mg ml À1 ), cisplatin (Faulding Pharmaceuticals, Warwickshire, UK) (0.156 -20 mg ml À1 ) and 5-FU (Faulding Pharmaceuticals) (0.156 -80 mg ml À1 ) was then added and the plate returned to the incubator for either 48 or 72 h. A background of media and a control of media and cells were also set up.
After 48 or 72 h of exposure to the chemotherapy agents, MTT (Sigma) was added and then incubated for a further 4 h at 371C. Using a scanning multi-well spectrophotometer (Dynatech MR5000, Dynatech Labarotaries Inc., Chantilly, VA, USA) measuring spectrophotometric absorbance at 570 nm, the plates were analysed using Biolinx 2.0 software (Biolinx 2.0, Dynatech Labarotaries Inc.). All experiments were repeated three times with six replicates per experiment. All subsequent cell work was based on the LD 10 and LD 50 concentrations of the chemotherapy agents at 48 h so that time was the only variable.

Quantifying 18 FDG uptake
Cells were seeded and incubated for 24 h, with control flasks were seeded at half the optimum seeding density so that cell density was similar in the control and treated flasks at the time of the 18 FDG uptake measurement. Control, LD 10 and LD 50 flasks were set up in triplicate and the corresponding concentrations of each chemotherapeutic agent and returned to the incubator for either 48 or 72 h. The incorporation of 18 FDG (obtained from the John Mallard PET Centre, Aberdeen) was determined by incubation of treated and control cells with 18 FDG (1 kBq ml À1 ) for 20 min at 371C followed by rapid washing with phosphate buffered saline (PBS) as described previously . 18 FDG uptake was expressed relative to protein content (milligram of cellular protein) and per treated flask.

Protein assay
Protein content was assessed by the bicinchoninic acid protein assay kit according to the manufacturers' instructions (Sigma).

Glucose transport
Initially the linear phase of [ 3 H]O-methylglucose ( 3 H-OMG) uptake, a measure of glucose transport (Cloherty et al, 2002), was determined by incubating cells with 3 H-OMG for 1, 2, 3, 5, 15 and 30 s. 3 H-OMG uptake in control AGS cells was very rapid, with the linear part of the time activity curve for 3 H-OMG uptake at 371C complete within 2 s of beginning incubation. Experiments were, therefore, conducted at 251C with an exposure to 3 H-OMG of 1 s.
Glucose transport rates were determined at both 48 and 72 h following the addition of required dose of chemotherapeutic agent, by incubation with media and 3 H-OMG (0.5 mCi ml À1 (specific activity 111GBq mmol À1 )), at 251C as described previously  except that the incubations were performed for 1 s. 3 H-OMGuptake was expressed in terms of protein content (milligram of cellular protein).

Hexokinase activity assay
AGS cells were set up and treated with chemotherapy as per 18 FDG uptake. Following the incubation for 48 or 72 h with chemotherapy the media was removed, and each flask was washed twice with ice-cold PBS. Cells were then trypsinised and centrifuged with the cell pellet washed a further two times with ice-cold PBS. The resulting cell pellet was then stored at À701C until the HK activity was assessed based on a modification to the methods by Miccoli et al (1996) as described previously .
Enzyme activity was expressed as mU mg À1 cellular protein using the extinction coefficient for NADPH of 6.3 Â 10 3 mol À1 cm À1 .
Flow cytometry DNA quantification AGS cells were set up and incubated in chemotherapy as previously described for 18 FDG uptake. After the required incubation period the media was discarded and the cells were harvested and cell cycle distribution was determined as described previously (Al-Saeedi et al, 2005).

Cell regrowth following exposure to chemotherapy
Cells were plated as for MTT assay and treated for 72 h with LD 50 and LD 10 doses of each chemotherapy agent. Following this, the media was removed and the cells were washed with warm (371C) PBS to remove any traces of residual chemotherapy. Fresh media (without chemotherapy) was then added and the plates were returned to the incubator. After 24, 48, 72, 96, 120 and 144 h incubation, MTT assays were performed to determine the latent cytotoxicity of the chemotherapeutic agents. Each experiment was repeated in triplicate with six replicates for each agent and time point.

Annexin V-PE flow cytometry
Annexin V flow cytometry was used to discriminate between intact cells, early apoptotic and late apoptotic or necrotic cells.
AGS cells were set up as per 18 FDG uptake. Control and cells treated for 72h with chemotherapy agents were detached by incubating the cells in non-enzymatic cell dissociation solution in PBS (Sigma) and added to fresh media. The cell concentration was then adjusted to 3 Â 10 5 cells ml À1 , and 1 ml of this cell suspension was then transferred to FACS tubes and centrifuged. The cell pellet was re-suspended in 1 ml of binding buffer (140 mM sodium chloride, 25 mM calcium chloride, 10 mM HEPES (N-(2-hydroxyethyl)piperazine-N 0 -(2-ethanesulphonic acid) hemisodium salt), 500 ml distilled water; Sigma). Following further centrifugation and removal of the supernatant, 15 ml of annexin V staining buffer (10 ml Via-Probe (7-N actinomycin D), 5 ml annexin V-PE) (BD Biosciences Pharmingen, Oxford, UK) was added to each cell sample. The samples were stored in the dark at room temperature for 15 min after which 400 ml of binding buffer was added and annexin V flow cytometry was performed within 1 h, analysing 10 000 cell events.
Annexin V flow cytometry was performed on a Becton Dickinson FACS Calibur (San Jose, CA, USA), and results analysed using CellQuest software (Becton Dickinson).

Statistics
Results are expressed as means, ±s.e.m.. 18 FDG, 3 H-OMG uptake and HK activity were expressed as a percentage of the control. Significance of difference between means was determined by using the paired t-test (Statistical Package for the Social Sciences V13.1, SPSS Inc., Chicago, IL, USA), with a P-value of o0.05 indicating statistical significance.

Cytotoxicity assay
Cytotoxic doses resulting in a 5 -10% decrease in cell number (LD 10 ) and LD 50 doses of chemotherapy after 48 h exposure were for cisplatin 0.156 and 5 mg ml À1 , 5-FU 0.156 and 20 mg ml À1 and epirubicin 0.0039 and 0.125 mg ml À1 respectively (Table 1).
Effect of chemotherapy on 18 FDG uptake 18 FDG uptake per untreated control flask (expressed as cpm per 20 min per flask) was 8714±1182 and 14 753±986 respectively after 48 and 72 h incubation periods. 18 FDG uptake, expressed as counts per milligram of cells per minute in untreated control flasks, was 783±94 and 984±35 (cpm per 20 min per mg protein) respectively following 48 and 72 h incubation periods.
All three chemotherapeutic agents caused a reduction in 18 FDG uptake per flask of treated cells ( Figure 1A and B), with the greatest reduction occurring following 72 h exposure to epirubicin. Upon 48 h exposure to LD 10 epirubicin, there was a non-significant reduction in 18 FDG uptake per flask (P ¼ 0.104), with a significant reduction upon exposure to LD 50 (P ¼ 0.006) compared with  (1) 69 (10) Cisplatin LD 10 5 (7) 5 (4) LD 50 56 (7) 77 (12) 5-Fluorouracil LD 10 9 (8) 13 (9) LD 50 43 (1) 55 (6) Results of MTT assay performed at 48 and 72 h. untreated controls. Both 48 h LD 10 and LD 50 exposure to cisplatin and 5-FU resulted in a significant reduction in 18 FDG uptake per flask (cisplatin, P ¼ 0.037 and P ¼ 0.001; 5-FU, P ¼ 0.001 and P ¼ 0.001). Compared with untreated controls there was a significant reduction in 18 FDG uptake per flask following 72 h LD 10 and LD 50 exposure to epirubicin (P ¼ 0.001 and P ¼ 0.001 respectively), cisplatin (P ¼ 0.001 and P ¼ 0.001 respectively) and 5-FU (P ¼ 0.001 and P ¼ 0.001 respectively). Exposure to all three chemotherapeutic agents resulted in a reduction in cellular 18 FDG uptake, with the greatest reduction (63%) occurring on exposure to LD 50 epirubicin after 72 h exposure (Figure 2A and B). This dose also produced a high level of cell death (69%). Compared with untreated controls there was a significant reduction in 18 FDG uptake following 72 h LD 10 (P ¼ 0.003) and both 48 and 72 h exposure to LD 50 epirubicin (P ¼ 0.001 and P ¼ 0.011 respectively), with a non-significant reduction in 18 FDG uptake following 48 h incubation at LD 10 levels (P ¼ 0.789). 18 FDG uptake was significantly decreased in cells exposed to LD 10 (P ¼ 0.01) and LD 50 (P ¼ 0.001) doses of 5-FU for 48 h exposure. Exposure to 5-FU for 72 h resulted in a significant decrease in 18 FDG uptake at LD 50 concentration (P ¼ 0.001) whilst there was little difference between control and LD 10 (P ¼ 0.839). Exposure for 48 h to cisplatin, LD 10 had little effect on 18 FDG uptake (P ¼ 0.636) in contrast to exposure of LD 50 , which resulted in a significant reduction in tracer uptake (P ¼ 0.001). After 72 h exposure to cisplatin there was little further reduction in 18 FDG cellular uptake compared with exposure to 48 h LD 50 .

Effect of chemotherapy on cell cycle
Exposure to LD 50 cisplatin and epirubicin resulted in cell cycle arrest in G 2 phase, with LD 10 doses resulting in G 1 arrest at both 48 and 72 h incubation periods. LD 10 dose of 5-FU resulted in S phase arrest, whilst the LD 50 caused G 1 arrest, irrespective of incubation periods ( Table 2).

Effect of chemotherapy on glucose transport
3 H-OMG cellular uptake rate was reduced upon exposure to epirubicin, cisplatin and 5-FU, with the greatest reduction (35%) in 3 H-OMG uptake resulting from 72 h exposure to LD 50 epirubicin ( Figure 3A and B). Forty-eight hours exposure to LD 50 epirubicin, cisplatin and 5-FU resulted in a significant reduction in cellular 3 H-OMG uptake compared to controls (P ¼ 0.037, P ¼ 0.005 and P ¼ 0.002 respectively) as did 72 h exposure (P ¼ 0.002, P ¼ 0.035 and P ¼ 0.001 respectively). LD 10 48 h exposure resulted in a nonsignificant decrease in 3 H-OMG uptake for epirubicin, cisplatin and 5-FU (P ¼ 0.064, P ¼ 0.539 and P ¼ 0.05 respectively). LD 10 exposure at 72 h caused a significant reduction in uptake for epirubicin (P ¼ 0.033) but not for cisplatin or 5-FU (P ¼ 0.304 and P ¼ 0.212 respectively).
Effect of chemotherapy on cellular HK activity LD 10 48 h exposure to epirubicin, cisplatin and 5-FU resulted in a non-significant increase in cellular HK activity ( Figure 4A and B) in comparison to controls (P ¼ 0.575, P ¼ 0.982 and P ¼ 0.465 respectively). Exposure to 48 h LD 50 , cisplatin and 5-FU also caused a non-significant increase in cellular HK activity (P ¼ 0.727 and P ¼ 0.282 respectively) whereas with epirubicin there was a non-significant reduction in HK activity (P ¼ 0.451).
Exposure to cisplatin and epirubicin for 72 h decreased HK activity, with the greatest reduction caused by exposure to LD 50 cisplatin (65%). Hexokinase activity was significantly reduced by exposure for 72 h to cisplatin and 5-FU (cisplatin P ¼ 0.047 and P ¼ 0.011, 5-FU P ¼ 0.016 and P ¼ 0.018 respectively for LD 10 and LD 50 ). Exposure for 72 h to epirubicin caused a significant reduction in HK activity with the LD 50 dose (P ¼ 0.02) but not the LD 10 dose (P ¼ 0.202).

Latent cytotoxicity of chemotherapeutic agents
Following 72 h exposure to both LD 10 and LD 50 of each individual chemotherapeutic agent, cells were washed and fresh media was added followed by further incubation for up to 6 days. Cells were able to recover from exposure to 5-FU and cisplatin when the drug was removed. However, epirubicin-treated cells did not recover even after 6 days of incubation in fresh media ( Figure 5A -C). Annexin V-PE flow cytometry revealed that AGS cells exposed to 72 h LD 50 epirubicin resulted in 69% cell death, 39% of surviving cells were annexin V-PE positive and 7-AAD negative, indicating this group of cells were in the early stages of apoptosis (Table 3).

DISCUSSION
Chemotherapy for tumours at or around the gastro-oesophageal junction is varied, with most regimes including one of epirubicin, cisplatin or 5-FU (Cunningham et al, 2006). All three chemotherapeutic agents caused a reduction in 18 FDG uptake by AGS cells with epirubicin having the greatest effect, followed by 5-FU then    cisplatin. Previous work on the AGS cell line using the same three chemotherapeutic agents and exposure times identified similar LD 50 concentrations of each agent, 5-FU 10 mg ml À1 , cisplatin 10 mg ml À1 and epirubicin 0.25 mg ml À1 (Couper and Park, 2003). In common with previous studies, exposure to epirubicin and cisplatin induced dose-dependent G 1 (LD 10 ) and G 2 (LD 50 ) cell cycle arrest Sorenson et al, 1990;Shapiro et al, 1998;Zoli et al, 2004). 5-FU exposure resulted in a build up of cells in S phase (LD 10 ) and G 1 (LD 50 ). Epirubicin, an anthracycline derivative of doxorubicin, exerts its anti-tumour effects via its action as a DNA intercalating agent and as an inhibitor of topoisomerase II (Cersosimo and Hong, 1986;Bartkowiak et al, 1992;Zoli et al, 2004). The arrest of AGS cells at higher concentrations of epirubicin may be related to peak activity of topoisomerases occurring during the G 2 phase (Chow and Ross, 1987). Exposure to cisplatin, an alkylating agent, results in the binding of cisplatin to DNA, forming cisplatin-DNA adducts which causes an alteration in the conformation of DNA leading to cell cycle arrest and apoptosis (Jordan and Carmo-Fonseca, 2000;Gonzalez et al, 2001;Wang et al, 2004). Cell cycle arrest following exposure to cisplatin occurs mainly within G 2 (Eastman, 1990;Sorenson et al, 1990), although this may be tumour type dependent (Sekiguchi et al, 1996;Shapiro et al, 1998). The main mechanism of action of 5-FU is related to its conversion to 5-fluoro-2 0 -deoxy-5 0 -monophosphate (via the pyrimidine pathway) leading to the inhibition of thymidylate synthase and hence DNA synthesis (Pinedo and Peters, 1988). Studies have revealed an increase S-phase fraction in tumour cells, caused by 5-FU Yamane et al, 1999;Park et al, 2004), including gastric carcinomas (Inada et al, 1997). Dittmann et al (2002), evaluating 18 FDG uptake in a squamous cell carcinoma oesophageal cell line, reported that 24 h incubation periods in concentrations of 5-FU and cisplatin, resulting in 22.8 and 60.6% cell death respectively, had no effect on cellular 18 FDG uptake, following a 24 h period of incubation in chemotherapy-free media prior to uptake experiments. Furthermore, after 24 h exposure to these same concentrations of drugs the S-phase fraction was elevated considerably, yet had no impact on 18 FDG uptake. Smith et al (2000) investigating tomudex, which is a more specific thymidylate synthase inhibitor than 5-FU, and oxaliplatin (a platinum agent similar to cisplatin) on a colonic tumour cell line found that exposure to tomudex for 24 and 48 h resulted in increasing levels of cellular 3 H-DG uptake with increasing exposure to the agent. This increase in uptake was paralleled with an increase in the S-phase fraction. The contrasts between these studies and ours may be in part explained by Yamane et al (1999), who revealed that although increasingly lengthy exposure to 5-FU resulted in S-phase accumulation of colorectal cancer cells with increased apoptosis, the Ki-67 labelling index decreased. Therefore, this S-phase accumulation is not proliferative but lethal. Exposure to oxaliplatin  resulted in decreased levels of cellular 3 H-DG uptake compared to controls with a varied cell cycle distribution. Although 18 FDG uptake in this study was found to be decreased after treatment with each agent, the effect on cell cycle was agent-specific suggesting that the changes in 18 FDG uptake are not cell cycle-specific, a finding previously reported by others (Higashi et al, 1993;Haberkorn et al, 1994).
Recently an analysis (Barros et al, 2005) based on the relationship between HK, glucose transport and intracellular glucose concentration shows that, at least in neuronal cells, the flux of glucose through the cell is dependent both on HK and glucose transport and that appreciable increases in flux require increases in both of these activities, whereas decreased flux can be brought about by a reduction in glucose transport or HK activity or both. Qualitatively, in our study, in each case where a decrease in 18 FDG incorporation is observed there is a corresponding decrease in glucose transport. By far the greatest decrease in 18 FDG incorporation is observed with the LD 50 dose of epirubicin for 72 h, which also marginally shows the greatest reduction in glucose transport. This treatment also causes an appreciable decrease in HK activity, which may augment the effect of glucose transport on 18 FDG incorporation. However, the lack of effect of the LD 10 dose of 5-FU for 72 h on 18 FDG incorporation or glucose transport, which caused a significant decrease in HK activity, suggests that glucose transport is the most important parameter for 18 FDG incorporation in this cell line. Further, regression analysis performed on pooled 48 and 72 h data from each treatment shows a strong correlation between the changes in FDG incorporation and glucose transport (t ¼ 0.863, n ¼ 12, Po0.001) but not between changes in FDG incorporation and HK activity (t ¼ 0.31, n ¼ 12, P40.1).
One possible reason for the closer association of glucose transport with 18 FDG incorporation compared with HK activity may be that the glucose transport assay uses intact cells so is a true measure of the glucose transport of the AGS cell, whereas the HK assay is performed in cell homogenates. Since HK activity in vivo is compartmentalised and highly regulated (Smith, 2000), cell breakage is likely to disrupt these regulatory systems.
We found that 18 FDG incorporation was consistently diminished by treatment with epirubicin, 5-FU or cisplatin. We did not see any evidence of increased incorporation. Although response to therapy is generally associated with decreased 18 FDG incorporation, a number of studies (Basu and Alavi, 2007) have reported that 18 FDG incorporation can increase in some responding tumours. In cases where this 'metabolic flare' phenomenon has been observed in breast tumours treated with anti-oestrogen treatment it has been attributed to the stimulatory effect of the anti-oestrogen at low blood concentrations (the situation when the patient begins therapy) on their cancer cells. A flare phenomenon occasionally found after radiotherapy generally corresponds with influx of inflammatory cells into the tumour (Kostakoglu and Goldsmith, 2004). Kubota et al (1992) studying 18 FDG uptake in a malignant murine model discovered that 18 FDG accumulated not only within the tumour cells, but also in the inflammatory components which appear with growth or tumour necrosis; however, the major source of 18 FDG was still tumour cells. Clearly neither of these situations is relevant to our treatment/model type.
Another type of hypermetabolism associated with 18 FDG has been reported in both solid tumours (Maruyama et al, 1999) and cell lines (Fujibayashi et al, 1997). This is likely to reflect biochemical changes within the cells in response to initial damage. We determined 18 FDG incorporation at times corresponding to Annexin V-PE FACS, 10 000 events counted per sample with results displayed as a percentage of valid counts (annexin V-PE and 7-AAD negative ¼ healthy cells; annexin V-PE positive and 7-AAD negative ¼ early apoptosis; annexin V-PE and 7-AAD positive ¼ necrosis or late apoptosis).
appreciable cell death so the initial response would have been complete.
Comparing 18 FDG incorporation after 48 and 72 h of treatment, when expressed relative to cellular protein, the decrease in 18 FDG incorporation is seen to plateau for treatment with cisplatin and 5-FU but not with epirubicin in which 18 FDG incorporation continues to decline. This may be explained by the high proportion (77%) of apoptotic cells in cell populations treated with LD 50 epirubicin for 72 h compared with cells exposed to cisplatin and 5-FU, suggesting that 18 FDG incorporation by apoptotic cells is decreased compared with viable cells. Following on from this, determination of the growth inhibitory effect of each agent by performing MTT measurements 6 days after treatment with each agent showed that AGS cells can recover from treatment with 5-FU and cisplatin but not after treatment with epirubicin. In assessing two chemotherapy regimens for gastro-oesophageal cancer, Couper and Park (2003) noted that AGS cells exposed to a combination of LD 50 of both cisplatin and 5-FU were able to recover following clearance of the chemotherapy. The addition of epirubicin to the combination of cisplatin and 5-FU resulted in a continual growth inhibitory effect (Couper and Park, 2003). The nature of Coupers' study meant that this effect may be due to the combined effect of three chemotherapeutic agents rather than specifically related to epirubicin. Engles et al treated MCF-7 breast carcinoma cells for 24 h with doxorubicin (an anthracycline similar to epirubicin) and 5-FU then re-incubated the cells in chemotherapy-free medium for a further 72 h. They found that cell number in populations treated with doxorubicin continued to decrease during the 72 h in doxorubicin-free medium (Engles et al, 2006), but addition of 5-FU-free media to MCF-7 cells treated for 24 h with 5-FU was associated with an increase in cell number indicative of recovery. It appears that the efficacy of epirubicin is associated with reduced cellular glycolytic rate (Zhou et al, 2002).
One of the limitations of our study is the extrapolation from in vitro to in vivo. Our studies are performed on well-perfused cells with a good nutrient and oxygen supply in an environment with neutral pH. Within solid tumours in vivo there are regions with compromised blood flow and consequent nutrient deprivation, lactic acid production and acidic pH. These are all factors that may influence 18 FDG incorporation. Burgman et al (2001) showed that induction of hypoxia resulted in increased 18 FDG incorporation by MCF-7 cells whilst HK activity is influenced by environmental pH (Miccoli et al, 1996). However, the region of tumour growth will have a good blood supply and is where most of the 18 FDG is likely to reach.
To simulate the uptake of 18 FDG within solid tumours, 18 FDG uptake was also expressed as activity per flask. The uptake of 18 FDG by detached cells could not be determined as steps to wash away non-incorporated 18 FDG would involve centrifugation, which would be a problem with detached cells as these are generally late dying/dead cells with fragile or damaged cell membranes. However, cells that were undergoing early apoptosis, for example, cells treated with epirubicin, were still attached and would be included in the analyses. Furthermore, in vivo, dead cells are rapidly removed by macrophages. So the contribution of dead cells in vivo is likely to be small.
In summary, treatment of gastric adenocarcinoma cells with cisplatin, 5-FU and epirubicin results in decreased 18 FDG incorporation. The greatest reduction in 18 FDG uptake per cell is induced by epirubicin. In contrast to cisplatin and 5-FU treated cells, epirubicin-treated cells did not recover when the drug was removed from the medium, corresponding with the annexin V-PE results, suggesting that the level of change in 18 FDG incorporation is predictive of tumour cell response. Each chemotherapeutic agent decreased glucose transport suggesting that glucose transport is the rate-limiting step for 18 FDG incorporation by AGS cells.