Abstract
In seven out of eight human bladder cell lines that were examined herein, growth was more dependent on the presence in the incubation medium of glucose rather than glutamine. The exception was the slowly growing RT4 cells that were more glutamine-dependent. Growth of all the cell lines was reduced by an inhibitor of 6-phosphofructo-2-kinase/2,6-bisphosphatase 3, namely 3-(3-pyridinyl)-1-(4-pyridinyl)-2-propen-1-one (3PO). Growth was also reduced by three compounds that reduce the conversion of glucose to lactate: namely 2-deoxyglucose, butyrate and dichloroacetate. Additive effects were seen when these molecules were combined with 3PO. Treatment of bladder cancer cells with phenformin resulted in growth inhibition that was frequently accompanied by increased glucose uptake and acidification of the medium that was blocked by co-incubation with 3PO. The actions of 3PO suggest that inhibitors of PFKB3 merit further investigation in the treatment of bladder cancer and they may be useful agents in combination with other drugs that inhibit cancer cell proliferation.
- Bladder cancer cells
- glycolysis
- growth
- PFKFB3 inhibitor
- butyrate
- 2-deoxyglucose
- dichloroacetate
- phenformin
Glucose and glutamine are major nutrients for cancer cells (1-3). The relative importance of these nutrients differs between cancer cell lines. Thus, glutamine was found to be the major energy source for HeLa cells (4), while for head and neck squamous carcinoma cells, glucose was the dominant energy source required for proliferation (5). There exists limited information on the importance of glutamine as a nutrient for bladder cancer cells (6). In the present study, we investigated the significance of glucose as an energy source for growth in a panel of human bladder cancer cell lines. The data suggested that glucose availability was critical for proliferation of most of the bladder cancer cells that were examined. This led to a study of potential inhibitors of glucose metabolism in bladder cancer cells as single agents and in combination treatment in the present investigation.
In cancer cells, glycolytic activity tends to be positively correlated with proliferation (7). The activity of phosphofructokinase 1 is a key site of regulation for glycolysis. Phosphofructokinase 1 is activated by fructose 2,6-bisphosphate the levels of which are controlled by bi-functional enzymes with phosphofructokinase 2 and fructose 2,6-bisphosphatase activities (PFKFBs). The high ratio of kinase to phosphatase activity of PFKFB3 and the increased expression of PFKFB3 in many cancer cells suggest a target for therapeutic intervention (8-11). Herein, we studied the action of 3-(3-pyridinyl)-1-(4-pyridinyl)-2-propen-1-one (3PO), an inhibitor of PFKFB3, as a single agent and in combination with other molecules that affect glycolysis and proliferation in eight human bladder cancer cell lines, The molecules studied were butyrate, 2-deoxyglucose, dichloroacetate and phenformin. One objective was to identify potential additive or synergistic effects on growth of bladder cancer cells.
Materials and Methods
Reagents. 3PO was purchased from EMD Millipore (Billerica, MA, USA). Sodium butyrate, 2-deoxyglucose, dichloroacetic acid and phenformin were obtained from Sigma-Aldrich (St. Louis, MO, USA).
Cell lines and determination of growth. Human bladder cancer cell lines, namely 5637, HT1197, HT1376, RT4, SW780, T24, TCCSUP and UM-UC-3, were obtained from the American Type Culture Collection (Rockville, MD, USA), and were incubated at 37°C in RPMI-1640 medium with 5% fetal calf serum. A total of 5×103 cells were plated in 0.2 ml of medium in 96-well plates. After allowing the cells to attach for 24 h, the medium was changed with addition of control medium or medium containing drugs. Cells were incubated for a further 72 h before growth determination and measurements on the medium. Cell growth was monitored by the increase in protein determined by staining with sulforhodamine B essentially as described by Vichai and Kirtikara (12).
pH Determination. pH determination with an electrode was found previously to correlate well with changes in the light absorbance at 560 nm reflecting changes in the pH indicator, phenol red (13, 14). Acidification of the medium has been found to correlate with lactate production by cancer cells (15) and provides a means of monitoring the impact of drugs on glycolytic activity.
Glucose assay. Glucose was assayed in the cell culture medium using Kit GAGO-20 from Sigma-Aldrich. This is a colorimetric procedure in which the oxidation of glucose is coupled with glucose oxidase and peroxidase to the oxidation of dianisidine.
Statistical evaluation. Data are presented as means and standard deviations for six or a greater number of determinations. Statistical significance of the results was determined by a two-tailed Student's t-test or by Dunnett's test for multiple comparisons. A probability of less than 5% was considered significant.
Results
Incubation of bladder cancer cells in medium lacking glucose blocked growth in seven out of the eight cell lines that were examined (Figure 1). The exception was the RT4 cell line in which there was inhibition of growth when glutamine was omitted from the medium. Overall growth was significantly reduced when glutamine was absent from the medium for six out of the eight cell lines.
There was significant inhibition of growth when most cell lines were incubated with either 10 μM 3PO or 1 mM butyrate but inhibition of growth was significant in all eight cell lines incubated with a combination of 3PO and butyrate (Figure 2).
Effects on acidification of the medium and uptake of glucose are more readily detected with the rapidly growing T24 and UM-UC-3 cells. This is illustrated in Figure 3, where a similar pattern of response to 3PO and butyrate is seen for the two parameters, with the greatest effect being observed in combination treatment.
The effects of combination treatment with two agents that affect glycolysis at different points in the metabolic sequence is shown in Figure 4. In rapidly growing UM-UC-3 cells and in the more slowly growing HT1376 and SW780 cells, there were inhibitory effects on growth when cells were incubated with 3PO and 2-deoxyglucose, the greatest inhibition being seen with a combined treatment.
Dichloroacetate appears to reduce lactate production by causing increased conversion of pyruvate to acetyl coenzyme A. Effects of dichloroacetate as a single agent and in combination with 3PO are shown in Figure 5. For the eight bladder cancer cell lines examined, the greatest inhibition of growth was observed with the combined treatment.
Phenformin differs from the other compounds studied in that it tends to increase glucose metabolism and increase acidification of the medium. This was more apparent with T24 cells than with UM-UC-3 cells, although the two cell lines had similar rapid growth rates (Figure 6). For both cell lines, there were significant inhibitions of growth when incubated with 25 or 50 μM phenformin (Figure 6C). The potential for 3PO and phenformin to exert additive inhibitory effects on the growth of bladder cancer cells is shown in Figure 7. The data in Figure 8 indicate that treatment with 3PO can reverse the increased acidification of the medium and increased glucose uptake seen when T24 cells are incubated with phenformin. The potential of phenformin to inhibit growth of bladder cancer cells was found to be enhanced when combined with another molecule that affects glucose metabolism, namely dichloroacetate, as shown in Figure 9.
Discussion
Bladder cancer is one of the more common types of human malignancy. Combination chemotherapy is frequently a part of treatment, particularly with combination of cisplatin and gemcitabine. Development of drug resistance is a problem (16) and there is a need for the identification of additional drug targets. Cancer metabolism offers such possibilities (17, 18). One target that is gaining increasing attention is enhanced glycolysis frequently seen in cancer and recognized since the work of Otto Warburg (19, 20). The identification of fructose 2,6-bisphosphate as a regulator of the key enzyme phosphofructokinase has led to increased attention being focused on the bi-functional PFKFB enzymes that control the level of fructose 2,6-bisphosphate. PFKFB3 may be of special interest because of its high ratio of kinase to phosphatase activity and increased expression in many types of cancer. A number of investigators have been seeking inhibitors of PFKFB3 that might exert selective toxicity on cancer cells (9, 21-24). 3PO was the first commercial inhibitor of PFKFB3 to become available. We chose to examine 3PO as a model compound in studies of combination treatments directed at glycolytic activity in colon cancer cells (25). Observation of growth-inhibitory effects prompted us to extend our studies to bladder cancer cells. Initially, we sought to determine if the glucose addiction, frequently seen in cancer, was a feature of bladder cancer cells. This was observed in a panel of eight human bladder cancer cell lines, in seven of which depletion of glucose was more critical than glutamine depletion. On the other hand, glutamine metabolism is of interest in the regulation of cancer proliferation (1, 3) and the present work indicates that glutamine can be important for the growth of bladder cancer cells.
Butyrate is a short-chain fatty acid that has been widely studied as a differentiating agent for several types of cancer cells. Its action as an inhibitor of histone deacetylase activity can influence the expression of many genes. This appears to include genes affecting glucose metabolism and suggests that histone deacetylase inhibitors may have a role to play in combination treatments directed at the inhibition of glycolysis. 2-Deoxyglucose is a more targeted agent, although not without effects beyond inhibition of glycolysis. As a single agent, it has not achieved a position in cancer therapy but in combination with other agents that affect glycolysis, it merits further investigation (13).
The bi-guanide metformin was found to inhibit the proliferation of bladder cancer cells in vitro and in vivo (26). However, these effects were seen at high concentrations, such as 5 mM, and use of metformin was not associated with a decreased incidence of bladder cancer in patients with type 2 diabetes (27). Phenformin is a bi-guanide that typically exhibits growth-inhibitory effects at lower concentrations than metformin and in the present study was generally effective at 50 μM and was additive with the action of 3PO. Combined treatment of cervical and breast cancer cells with metformin and dichloroacetate has been reported to enhance cytotoxicity of metformin (28). We examined the combined action of phenformin and dichloroacetate on bladder cancer cells and observed additive effects on cell growth. One advantage of this approach is that is that it may prevent lactic acidosis caused by phenformin that resulted in the withdrawal of approval for treatment of diabetes with phenformin in the United States. Kankotia and Stacpole have reviewed the anticancer effects of dichloroacetate and were sufficiently encouraged by the data to recommend more extensive investigation of the compound (29).
3PO may be regarded as a lead compound in studying the impact of PFKFB3 inhibition in controlling proliferation of cancer cells. Structurally related molecules can be effective at lower concentrations than with 3PO (10). There is evidence that PFKFB4 can be elevated in bladder cancer and may be a marker of cancer progression (30); hence more widely acting PFKFB inhibitors may be of interest (31). Bladder cancer is heterogeneous and presents many targets for therapeutic intervention (32). The data presented here suggest that glucose metabolism is one of the targets to be investigated.
Acknowledgements
This research was supported by the Alma Toorock Memorial for Cancer Research.
- Received July 15, 2015.
- Revision received September 8, 2015.
- Accepted September 16, 2015.
- Copyright© 2015 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved