Introduction
Pancreatic cancer is a globally lethal human disease, with an overall 5-year survival rate of <4% (1), and a median survival period of 4–6 months (2,3). It is considered the fourth leading cause of cancer mortality in males and females (4). The nucleoside analog of cytidine 5-fluorouracil (5-Fu) is widely used in the treatment of advanced gastrointestinal cancer, including pancreatic cancer (5–7).
However, only few patients benefit from 5-Fu-based chemotherapy. Intrinsic or acquired resistance to chemotherapy is a leading cause of treatment failure and short survival time (8,9). The reasons for the insensitivity of pancreatic cancer cells to chemotherapy and the molecular mechanisms that enable pancreatic cancer cells to escape the cytotoxic effects have yet to be determined (10–12).
Abnormal biochemical characteristics associated with pancreatic cancer cells include the increased utilization of glucose (13). Increased proliferation depends on abnormal glucose metabolism for the generation of ATP as a main source of energy supply as most cancer cells lack oxidative phosphorylation. This phenomenon is known as the Warburg effect (14–18). This metabolic alteration is frequently observed in cancer cells of various tissue origins, thus targeting the glycolytic pathway may preferentially kill the malignant cancer cells but spare normal cells.
Previously, we demonstrated that PI3K-Akt activated by NGF-TrkA signaling was involved in the resistance to chemotherapy (19). Akt may be considered as the ‘Warberg gene’ (20), which is closely associated with tumor glycolysis and glucose utilization. Since pancreatic cancer cells demonstrate increased utilization of glucose, it is crucial to target glycolysis metabolic pathway for the treatment of pancreatic cancer.
To examine whether high glucose plays a role in the resistance to 5-Fu and whether the inhibition of glycolysis using glycolysis inhibitor 2-deoxy-D-glucose (2-DG) results in enhanced sensitivity to 5-Fu, we investigated cell viability by 5-Fu treatment on different concentrations of glucose in AsPC-1 and Panc-1 pancreatic cancer cells. Additionally, we investigated whether 2-DG is able to reverse high glucose-induced 5-Fu resistance via PI3K-Akt signaling.
Materials and methods
Reagents
5-Fu, dimethyl sulfoxide (DMSO), 2-DG, glucose and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were purchased from Sigma-Aldrich (St. Louis, MO, USA). RIPA buffer and PMSF were purchased from Beyotime (Haimen, Jiangsu, China). Anti-p-Akt and PI3K inhibitor LY294002 were purchased from Abcam (Cambridge, MA, USA). Anti-β-actin antibody was from Abnova (Taiwan, China).
Cell culture
Human pancreatic cancer AsPC-1 and Panc-1 cells were purchased from the Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China). The cells were grown in RPMI-1640 medium (Gibco, Carlsbad, CA, USA) supplemented with 10% heat-inactivated FBS (Hyclone, Logan, UT, USA), penicillin 100 U/ml and streptomycin 100 μg/ml (Gibco). The cultures were maintained at 37ºC in a 5% CO2 incubator.
Cell growth inhibition assay
Cell viability was assessed via an MTT assay. ASPC-1 and Panc-11 cells were seeded (3,000/well) in 96-well plates for 24 h. Media containing 5-Fu, 2-DG, LY294002 or control medium were added and incubated for the indicated times at 37ºC. MTT (0.5 mg/ml in PBS) was added to each well and incubated for 4 h at 37ºC. The media were then discarded and 100 μl DMSO was added. Following agitation for 10 min on an eppendorf shaker, absorbance was read at 550 nm on a scanning microtiter. Data were expressed relative to the untreated group, which was set as 100%.
Western blot analysis
Cells were lysed with modified RIPA buffer (50 mM Tris, 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS) containing 25 μg/ml leupeptin, 1 mM sodium orthovanadate, 2 mM EDTA, and 1 mM PMSF. The protein concentration was determined using a BCA method (Beyotime). Twenty micrograms of proteins per sample were loaded onto 8% SDS-polyacrylamide gel, electrophoresed, and blotted onto PVDF membrane. Proteins were probed with the primary antibody overnight at 4ºC and secondary antibody at room temperature for 1 h. Immunoreactivity was detected by the ECL system (Xi’an Jiaotong University, China) and normalized to β-actin.
Statistical analysis
Data were analyzed by SPSS 13.0 using t-test. P<0.05 was considered statistically significant.
Results
High-glucose microenvironments alleviated 5-Fu-induced cell growth inhibition
To investigate the influence of glucose levels on resist to 5-Fu, cells were incubated in a series of gradually increasing glucose concentrations for 72 h with 1 mM of 5-Fu.
Increased cell growth of AsPC-1 and Panc-1 cells treated with 5-Fu was observed in response to the glucose concentrations ranging from 5.6 to 25 mM. In AsPC-1 and Panc-1 cells, the cell viability rate was increased in a dose-dependent manner at glucose concentrations of 5.6 mM (as a control) to 25 mM, at 72 h, respectively (P<0.05) (Fig. 1). In comparison with parental ASPC-1 and Panc-1, incubation with 5-Fu for 72 h at the glucose concentration of 5.6 mM decreased the cell number to 28 and 31%, respectively (P<0.05). High-glucose microenvironments showed a marked effect on the growth of AsPC-1 and Panc-1 cells. At the glucose concentration of 25 mM, 5-Fu decreased the cell number to 55 and 72%, respectively (P<0.05). The cytotoxic effect of 5-Fu reduced glucose in a concentration-dependent manner.
2-DG enhanced cytotoxic effects of 5-Fu in high-glucose microenvironments
Several studies demonstrated that 2-DG induces cell growth inhibition and death in pancreatic cancer cells by interfering with glucose metabolism. We hypothesized that the enhanced resistance to 5-Fu in glucose microenvironments may be blocked by the anti-glucose metabolism treatment of 2-DG. To test this, 2-DG (10 mM) was used to interfere with cell glucose metabolism and detect the sensitivity of the two cell responses to 5-Fu treatment at the glucose concentration of 25 mM. Growth of AsPC-1 and Panc-1 was inhibited by incubation with 5-Fu or 2-DG alone in a time-dependent manner (P<0.05). Treatment of tumor cells with 0.5 mM 5-Fu combined with 5 mM 2-DG revealed a marked decreased in cell growth compared with 5-Fu or 2-DG alone (P<0.05), leading to a decrease of 54% in AsPC-1 and 52% in Panc-1 at 72 h of incubation (Fig. 2).
2-DG enhanced 5-Fu cytotoxic effect depending on PI3K signaling
Treatment of AsPC-1 and Panc-1 cells with 10 mM 2-DG resulted in ~38 and 41% inhibition of cell growth, respectively, in a time-dependent manner. Three concentrations (6.1, 7.8 and 25 mM) of glucose were selected to detect the expression of Akt, also known as the Wurberg gene and to determine whether it can be inhibited by 2-DG (Fig. 3A). As cells incubated with higher concentrations of glucose exhibited increased activity of p-Akt, we examined whether the upregulation of Akt by glucose is important in 2-DG-induced cell growth. Consequently, we treated cells with LY294002 and found that LY294002 significantly attenuated the death of 2-DG-induced cells (Fig. 3B and C). The combination of 5-Fu and 2-DG did not exhibit any significance compared with 5-Fu alone. Although 2-DG reduced the expression of Akt, blocking PI3K/Akt signaling using LY294002 did not enhance the inhibition of 5-Fu.
Discussion
2-DG is a glucose analog and acts as a competitive inhibitor of glucose metabolism, causing a depletion of cellular ATP (21), leading to blockage of cell cycle progression (22), and inducing cell death (23). In addition, 2-DG inhibits protein glycosylation, and induces the accumulation of misfolded proteins in the endoplasmic reticulum, leading to endoplasmic reticulum stress response and constant cell apoptosis (24). 2-DG has been proven to be an anti-cancer drug for a variety of cancer cells and increases the efficacy of radiotherapy and chemotherapy agents such as adriamycin and paclitaxel (25,26).
In the present study, we used glucose as a model to enhance cell resistance to the widely used anti-pancreatic cancer agent 5-Fu in AsPC-1 and Panc-1 pancreatic cancer cells. The results demonstrated that active PI3K-Akt by high-glucose concentrations decreased the antitumor effect of 5-Fu, which is in agreement with previous studies (19,27–31). Of note, 2-DG significantly reversed the resistance to 5-Fu in 25 mM of glucose. Our data demonstrated that PI3K-Akt is required for 2-DG-induced cell growth inhibition. Similarly, enhanced proliferation due to NGF-TrkA signaling or loss of PTEN makes cells more sensitive to 2-DG (32,33).
Our results emphasize that an increase in blood sugar as a result of diabetes, which is closely associated with pancreatic cancer may increase the risk of cancer proliferation and resistance to chemotherapy (34–37). Controlling blood sugar levels is significant in the therapy of pancreatic cancer. However, this resistance can be alleviated by glycolysis inhibition using 2-DG. Our results suggest that targeting glucose glycolysis is a viable approach for the development of anti-cancer drugs, particularly for patients experiencing difficulty in reducing blood sugar levels.
Activation of the PI3K/AKT pathway has been implicated in a variety of tumors (38,39), mediating tumor growth, and exhibiting resistance to apoptosis and chemotherapy. PI3K/AKT pathway inhibition leads to a wide spectrum of direct effects including cell cycle arrest, induction of autophagy, sensitization to chemotherapeutics, inhibition of metastasis as well as cell differentiation and death (40,41). Results of the present study have shown that glucose and 2-DG were involved in the regulation of PI3K/Akt signaling, and this may explain the effect of aberrant glucose metabolism in pancreatic cancer, and highlight the marked efficiency of 2-DG in high-glucose microenvironments and the significance of controlling the blood sugar of pancreatic cancer patients, particularly those with diabetes. However, blocking PI3K/Akt did not provide an adequate explanation for the entire mechanism of 2-DG, or the effect of 5-Fu being greatly enhanced by using LY294002. Thus additional studies should be conducted to further elucidate the mechanism involved.
In conclusion, the results of the present study demonstrated that the effect of 5-Fu-based chemotherapy on pancreatic cancer is significantly reduced by high glucose, although this effect can be reversed by 2-DG. It is therefore crucial for pancreatic cancer patients to control blood sugar levels in order to fully benefit from chemotherapy.