Combined 18F-FDG PET/CT imaging and a gastric orthotopic xenograft model in nude mice are used to evaluate the efficacy of glycolysis-targeted therapy

  • Authors:
    • Ting-An Wang
    • Shu-Lin Xian
    • Xing-Yu Guo
    • Xiao-Dong Zhang
    • Yun-Fei Lu
  • View Affiliations

  • Published online on: October 31, 2017     https://doi.org/10.3892/or.2017.6060
  • Pages: 271-279
Metrics: Total Views: 0 (Spandidos Publications: | PMC Statistics: )
Total PDF Downloads: 0 (Spandidos Publications: | PMC Statistics: )


Abstract

As discovered by Warburg 80 years ago most malignant cells rely more on glycolysis than normal cells. The high rate of glycolysis provides faster ATP production and greater lactic acid for tumor proliferation and invasion, thus indicating a potential target in anticancer therapy. Our previous studies demonstrated that 3-bromopyruvate (3-BrPA) and sodium citrate (SCT) inhibited tumor cell proliferation in vitro. However, the underlying mechanisms still warrant further investigation. In the present study, we employed the human SGC-7901 gastric cancer cell line, built an orthotopic xenograft model in nude mice, examined the treatment response by 18F-FDG PET/CT and investigated the mechanisms of 3-BrPA and SCT in vivo. Our results demonstrated that glycolysis and tumor growth were inhibited by intraperitoneal injection of 3-BrPA and SCT, which were imaged using an 18F-FDG PET/CT scanner. In addition, apoptosis induced by 3-BrPA and SCT was initiated by the upregulation of Bax and downregulation of Bcl-2, which promote cytochrome c release and subsequently activate caspase-9 and -3, and ultimately execute mitochondria-mediated apoptosis. Furthermore, apoptosis was also modulated by the generation of ROS and inhibition of survivin. Accordingly, 3-BrPA and SCT can inhibit glycolysis and induce gastric cancer apoptosis through the mitochondrial caspase-dependent pathway.

Introduction

Despite advances in surgical techniques, adjuvant chemotherapy, radiotherapy and immune therapy in the past decades, cancer is still the second leading cause of death worldwide alongside heart diseases (1). In particular, gastric cancer (GC), with the highest prevalence in Eastern Asia (including Japan, Korea and China) worldwide, has a 5-year survival rate of less than 30% (1,2). Therefore, the discovery of novel and more effective therapeutic targets against cancer appears to be of central importance.

In 1956, Otto Warburg observed that malignant cells undergo a higher rate of glycolysis for energy in contrast to healthy cells (3). Based on this characteristic of cancer cells, 2′-[18F]-fluoro-2′-deoxy-D-glucose positron emission tomography (18F-FDG PET) has been extensively used to diagnose and assess various cancers including GC, and the changes in 18F-FDG uptake can also reflect early treatment response (4,5). In addition, this glycolytic metabolic characteristic of cancer cells also suggests that targeting glycometabolism may be an effective way to selectively inhibit cancer cells (6). Moreover, this dysregulated metabolism has been revealed to promote drug resistance in malignant cells (7).

Recently, a number of molecules have been discovered to suppress glycolysis in cancer cells. Various representative drugs are iodoacetic acid (IAA), dichloroacetate (DCA), 2-deoxyglucose and 3-bromopyruvate (3-BrPA). Previous studies from Pedersen and our team revealed that 3-BrPA decreased glycolysis by suppressing mitochondrial hexokinase activity (8,9). Similarily, sodium citrate (SCT), a member of the mitochondrial tricarboxylic acid cycle, has also been revealed to inhibit medullary thyroid cancer, leucocythemia, malignant pleural mesothelioma and ovarian carcinoma growth (1013).

It is generally known that the potential anticancer ability of a new therapeutic approach is often evaluated by activation of tumor cell apoptosis. There are 2 major signaling pathways that induce apoptosis: the extrinsic (death receptor) pathway mediated by procaspase-8, and the intrinsic (mitochondrial) pathway which is triggered by the disruption of the mitochondrial transmembrane potential, release of apoptogenic proteins such as cytochrome c (Cyt-C) into the cytoplasm and subsequent activation of caspase-9 (1416). The 2 signaling pathways converge at the activation of caspase-3, a key mediator in the execution apoptosis (17). Furthermore, the proteins in the Bcl-2 family such as pro-apoptotic Bax and anti-apoptotic Bcl-2, modulate the mitochondrial membrane potential thus regulating apoptotic signaling (18). In addition, as a member of the IAP family, survivin is abundantly expressed in the majority of human cancers, and is associated with tumor proliferation and treatment resistance (19). Survivin inhibits the activation of caspases and is a target of anticancer drugs (20,21). Moreover, reactive oxygen species (ROS) generated in and around mitochondria, has been indicated to induce uncontrolled oxidative stress and subsequent cell apoptosis (22).

The application of tumor animal models is extremely important in pre-clinical studies of oncology (23), and traditional ectopic models were restricted in their poor representation of the pathophysiological milieu around tumors (24). Thus, it is necessary to create practicable orthotopic xenograft tumor models that mimic the biological characteristics of human tumors in order to discover successful therapeutic strategies (25).

The results from our previous studies indicated that the antitumor mechanisms of 3-BrPA and SCT warrant further investigation (9,13). In the present study, we created a GC orthotopic xenograft model in nude mice and intraperitoneally injected 3-BrPA and SCT. The antitumor efficacy of 3-BrPA and SCT was detected using a micro-PET/CT scanner. We also explored the underlying mechanisms in the inhibition of tumors induced by 3-BrPA and SCT. In the present study the results demonstrated that the antitumor effect was associated with the generation of ROS, upregulation of Bax, downregulation of Bcl-2 and survivin, release of Cyt-C and activation of caspase-3 and −9 cascades.

Materials and methods

Cell line and reagents

Gastric carcinoma cell line SGC-7901 was purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). The cell line was maintained in RPMI-1640 medium supplemented with 10% fetal calf serum (Gibco, Carlsbad, CA, USA), 100 U/ml penicillin and 100 µg/ml streptomycin, in a humidified atmosphere with 5% CO2 at 37°C.

3-BrPA, SCT and 5-fluorouracil (5-FU) were purchased from Sigma-Aldrich (St. Louis, MO, USA), dissolved in medium and phosphate-buffered saline (PBS) to create working solutions and filtered and sterilized prior to treatment.

Cell proliferation assay

In order to determine the sensitivity of the SGC-7901 cell line to 3-BrPA, SCT or 5-FU, ~2,000 cells were seeded into 96-well plates and exposed to varying concentrations of the drugs for 24 and 48 h. Then, the cells were incubated with 10 µl of 5 mg/ml 3-(4,5-demethylthiazol-2-yl)-2,5-diphenyltetrazonium bromide (MTT) for a further 4-h incubation at 37°C. Dimethyl sulfoxide (DMSO) was added to each well, and was shaken for 30 sec. The absorbance at 490 nm was quantified using a microplate reader (Bio-Rad, Hercules, CA, USA).

Hochest 33258 staining

To detect apoptosis in vitro, SGC-7901 cancer cells were grown in 6-well plates and treated with 3-BrPA, SCT and 5-FU for 24 h, then stained with Hochest 33258 (Beyotime Biotechnology, Jiangsu, China), and visualized under a fluorescence microscope (Olympus, Tokyo, Japan) at an excitation wavelength of 350 nm and an emission wavelength of 460 nm.

Detection of intracellular ROS level

The production of peroxides was assessed using the ROS assay kit (Beyotime, Haimen, China). Briefly, SGC-7901 cancer cells were treated with 3-BrPA, SCT and 5-FU for 4 h, incubated with 10 µM DCFH-DA in serum-free medium at 37°C for 20 min. Subsequently, the cells were imaged with a laser scanning confocal microscope (Nikon A1; Nikon Corporation, Tokyo, Japan) at excitation and emission wavelengths of 488 and 525 nm, respectively. The fluorescence intensity was regarded as the generation of ROS.

Establishment of a gastric carcinoma orthotopic xenograft model

Four-week-old female BALB/c athymic nude mice, weighing 20±2 g, were obtained from the Animal Experimental Center of Guangxi Medical University (Guangxi, China) and fed under specific pathogen-free conditions. All experimental procedures were conducted in accordance with the internationally recognized guidelines for conduct and animal welfare.

Mice were subcutaneously injected with 200 µl of suspension (2×106 cells/ml) of the SGC-7901 cell line in the back of axillary regions and sacrificed by cervical dislocation after 14 days when the palpable tumor diameter reached 1.0 cm. Tumor tissues were stripped and minced to 1–2 mm3, and then transplanted into the next new group of mice for 6 sequential generations. The sixth subcutaneously transplanted tumor was used as the source of orthotopic transplantation.

Nude mice were anesthetized with sodium pentobarbital (45 mg/kg of body weight) by intraperitoneal injection, and an incision was made to carefully expose the stomach. The seromuscular layer of greater curvature was punctured with a needle to form a local concave niche where a tumor mass of ~1.0 mm3 with good conditioning was imbedded and then the surface and greater omentum was covered with medical OB glue.

After transplantion for 2 weeks, 96 mice were randomly divided into the following 8 groups (n=12): control group (PBS, 10 ml/kg), 5-FU group (5-FU, 10 mg/kg), 3-BrPA low-dose group (3-BrPA-L, 1.85 mg/kg), 3-BrPA medium-dose group (3-BrPA-M, 2.23 mg/kg), 3-BrPA high-dose group (3-BrPA-H, 2.67 mg/kg), SCT low-dose group (SCT-L, 7.5 mg/kg), SCT medium-dose group (SCT-M, 15 mg/kg), and SCT high-dose group (SCT-H, 30 mg/kg). Nude mice were daily intraperitoneally injected with corresponding drugs or PBS (10 ml/kg), respectively. For 4 weeks the physical, stool and abdominal conditions of nude mice were observed throughout this experiment. After a 4-week treatment, half of the nude mice in each group were imaged with a micro-PET/CT scanner and then sacrificed by cervical dislocation. The tumor tissues and organs were stripped for subsequent studies. The remaining mice were continually treated until their death in order to evaluate the life prolonging rate.

Small-animal 18F-FDG PET/CT scanning

A small-animal PET/CT scanner (Inveon; Siemens Medical Solutions, Knoxville, TN, USA) was used for in vivo imaging. The SGC-7901 tumor-bearing mice were anesthetized with isoflurane (2% in 98% oxygen), kept at 38°C and the caudal vein was injected with 18F-FDG (5.55 MBp, 150 µCi) 40 min prior to imaging. A PET scan for 15 min was conducted on the mice followed by a 5 min CT scan. Three-dimensional regions of interest (ROI) were drawn around the gastric tumors and the standardized uptake value was measured as the percentage of injected radioactivity dose/gram (% ID/g).

Hematoxylin and eosin and immunohistochemistry (IHC) staining

The harvested tumor tissues, livers and kidneys were embedded in paraffin, sectioned at 5-µm thickness and stained with hematoxylin and eosin (H&E) for examination by light microscopy.

In addition, immunohistochemical (IHC) staining was performed using antibodies for cleaved caspase-9 (1:100 dilution) and cleaved caspase-3 (1:400 dilution) (both from Cell Signaling Technology, Danvers, MA, USA). The expression of cleaved caspase-9 and cleaved caspase-3 were determined based on the cytoplasmic and nuclear staining. The results were scored according to the mean density (MD) by Image-Pro Plus 6.0 and evaluated by 2 pathologists.

Lactate, ATP and glycolytic enzyme assay

To detect the inhibitory effect on glycolysis of 3-BrPA and SCT in vivo, the lactate production, ATP content and the glycolytic enzyme activity (HK, PFK-1 and PK) were assessed using assay kits (Jiancheng, Nanjing, China). According to the manufacturer's instructions, the absorbance at 340 nm was assessed spectrophotometrically (UV-2450; Shimadzu, Kyoto, Japan) and the results were calibrated with cellular protein concentration.

Western blot analysis

Total proteins were extracted from orthotopic tumors, electrophoresed in sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), and then transferred onto a nitrocellulose filter membrane (Millipore, Billerica, MA, USA). Non-specific binding was blocked with 5% skimmed milk for 2 h at room temperature and incubation followed with specific primary antibodies at 4°C overnight. The primary antibodies used were rabbit antibodies against Bax, Bcl-2, Cyt-C, survivin and GAPDH (1:1,000 dilution; Cell Signaling Technology). The anti-rabbit IgG DyLight 800-conjugated antibody was incubated at room temperature for 90 min (1:1,000 dilution; Cell Signaling Technology). The protein bands were visualized and quantified using the Odyssey system (LI-COR Biosciences, Lincoln, NE, USA).

Results

Drug sensitivity of cell lines

As shown in Table I, the IC50 values of 3-BrPA, SCT or 5-FU in the SGC-7901 GC cell line were 10.23±2.37 µg/ml and 7.19±1.05 mg/ml at 24 h, 6.15±1.24 µg/ml and 4.12±0.87 mg/ml at 48 h, 0.38±0.05 and 0.25±0.03 mmol/l at 48 h respectively.

Table I.

IC50 values of 3-BrPA, SCT and 5-FU in the SGC-7901 cancer cell line in vitro.

Table I.

IC50 values of 3-BrPA, SCT and 5-FU in the SGC-7901 cancer cell line in vitro.

Groups3-BrPA (µg/ml)SCT (mg/ml)5-FU (mmol/l)
24 h10.23±2.377.19±1.050.38±0.05
48 h6.15±1.244.12±0.870.25±0.03

[i] The results are expressed as the mean ± SD. 3-BrPA, 3-bromopyruvate; SCT, sodium citrate, 5-FU, 5-fluorouracil.

Apoptosis is assessed by Hochest 33258 staining

The apoptotic cells, characterized by chromatin condensation and fragmentation, were observed after exposure to 3-BrPA and SCT for 24 h (Fig. 1A and C). The percentage of apoptotic cells increased along with the concentrations of 3-BrPA and SCT. The results strongly indicated that 3-BrPA and SCT induced SGC-7901 cell apoptosis in vitro.

ROS is generated after 3-BrPA and SCT treatment

Intracellular ROS generation was evaluated using intracellular peroxide-dependent oxidation of DCFH-DA to form fluorescent DCF and detected with laser scanning confocal microscope after treatment with 3-BrPA and SCT for 4 h (Fig. 1B). ROS production was significantly increased upon treatment with 3-BrPA, SCT and 5-FU compared with the control (Fig. 1D; p<0.05). Furthermore, the fluorescence intensity increased along with the dose of 3-BrPA and SCT.

Effect of 3-BrPA and SCT on gastric orthotopic xenograft tumors

Gastric orthotopic xenograft models were successfully built (Fig. 2A and B), and the effect of 3-BrPA and SCT on tumor growth and survival time in mice were investigated. The orthotopic xenograft tumors were identified by touch through the abdominal wall 10 days after transplantation and the tumor formation rate was ~95% and the mortality rate was 4%. The mice administered by intraperitoneal injection with 3-BrPA and SCT exhibited tumor growth inhibition compared with the control group (Table II) (p<0.05). Moreover, as shown in Fig. 2D, the survival time of mice increased in the 3-BrPA and SCT groups (p<0.05). The results demonstrated that 3-BrPA and SCT suppressed tumor growth and prolonged tumor-bearing mice survival time.

Table II.

Results of nude mice weights, tumor volumes, tumor weights, and the inhibitory rate of tumors (%).

Table II.

Results of nude mice weights, tumor volumes, tumor weights, and the inhibitory rate of tumors (%).

GroupNude mice (n)Tumor volume (mm3)Tumor weight (g)Inhibitory rate of volume (%)Inhibitory rate of weight (%)
3-BrPA-L12810.41±72.510.80±0.05 29.70±6.29a,b 30.23±3.43a,b
3-BrPA-M12730.08±60.100.70±0.04 36.67±5.21a,b 39.43±5.09a,b
3-BrPA-H12643.03±77.760.62±0.05 44.22±6.75a 46.86±5.96a
SCT-L12799.78±64.790.79±0.06 30.62±5.62a,b 29.18±3.14a,b
SCT-M12708.60±86.930.69±0.07 38.53±7.54a,b 38.02±4.65a,b
SCT-H12615.72±57.000.60±0.03 46.59±4.94a 45.62±4.82a
5-FU12605.48±61.530.60±0.05 47.48±5.34a 47.04±5.07a
Control121152.77±88.991.13±0.080.00b0.00b

{ label (or @symbol) needed for fn[@id='tfn2-or-39-01-0271'] } The growth of orthotopic xenograft tumors in nude mice was markedly inhibited with 3-BrPA and SCT treatment (aP<0.05). Results are presented as the mean ± standard deviation (n=12).

a p<0.05 vs. control

b p<0.05 vs. 5-FU group. 3-BrPA, 3-bromopyruvate; SCT, sodium citrate, 5-FU, 5-fluorouracil.

Histology assessment

In order to determine whether 3-BrPA and SCT had a toxic effect on mice, the histopathological changes in the liver and kidneys were observed by H&E staining. As shown in Fig. 2C, histological examination did not reveal any apparent abnormalities, indicating the safety of the 3-BrPA and SCT doses used in the present study. However, slight vacuolar changes and spotty necrosis in hepatocytes, hydropic changes and leukocyte infiltration in kidney cells were observed in the 5-FU-treated mice.

3-BrPA and SCT suppresses glycolytic activity in tumors as determined by micro-PET/CT imaging

PET/CT with 18F-FDG is a non-invasive approach to detect the glycolytic activity, and the radiotracer accumulation (% ID/g) in tumor areas, reflecting the intensity of tumor glucose metabolism. In the present study, we observed a significantly decreased radioactivity uptake in the mice treated with 3-BrPA and SCT (Fig. 3) (p<0.05), which may have resulted from the inhibition of PFK-1 and HK activities. Notably, a decrease of radiotracer accumulation was also observed in the 5-FU-treated group, which may be associated with the decrease of HK activity.

3-BrPA and SCT activate caspase cascades

Activation of caspase-3 and −9 is a key downstream event involved in the initiation and execution of apoptosis. From the IHC analysis (Fig. 4), the tumors from mice treated with 3-BrPA and SCT exhibited increased levels of cleaved caspase-3 and −9 in a dose-dependent manner, suggesting that the mechanism of apoptosis induced by 3-BrPA and SCT is related to caspase activation (p<0.05). Similarly, a significant increase in the expression of cleaved caspase-3 and −9 was also found in tumors with the 5-FU treatment.

Effects of 3-BrPA and SCT on Bcl-2, Cyt-C, Bax and survivin expression

To further investigate the possible underlying mechanism responsible for executing 3-BrPA and SCT induced apoptosis, we analyzed the expression of Bcl-2, Bax, Cyt-C and survivin using western blot analysis (Fig. 5A). The 3-BrPA and SCT-treated groups exhibited an increased expression of pro-apoptotic factors, Bax and Cyt-C, and a decreased expression of anti-apoptotic factors, Bcl-2 and survivin, in a concentration-dependent manner (p<0.05). However, with 5-FU treatment, Bax and Cyt-C were also upregulated while Bcl-2 and survivin were downregulated (p<0.05). These results from western blot and IHC analyses indicated that the mechanism of induced apoptosis by 3-BrPA and SCT may be mediated by suppressing survivin and activating the mitochondria-dependent pathway.

3-BrPA and SCT inhibit the activities of glycolytic enzymes to decrease lactate and ATP production

To further explore the potential mechanisms involved in the regulation of glucose metabolism of 3-BrPA and SCT in vivo, the activities of glycolytic enzymes (PK, HK and PFK-1), lactate and ATP production in tumor tissues was assessed (Fig. 5B and C). The data revealed that lactate and ATP production was decreased in the 3-BrPA-treated group by suppression of HK activity, and it was also decreased in the SCT-treated group by inhibition of PFK-1 activity. Lactate and ATP production in the 5-FU-treated group was significantly lower in contrast to the PBS group (p<0.05). Moreover, the activity of HK and PFK-1 was suppressed by 3-BrPA and SCT in a concentration-dependent manner, respectively.

Discussion

As discovered by Warburg 80 years ago, most malignant cells preferentially utilize glycolysis rather than oxidative phosphorylation for energy production even in the presence of oxygen (3) although the energy generation of glycolysis is far less efficient than oxidative phosphorylation (2 ATPs/glucose in glycolysis, but 36 ATPs from oxidative phosphorylation). However, increased glycolysis not only provides faster ATP production, but also greater lactic acid for tumor cell proliferation and invasion and may promote their resistance to chemoradiotherapy (3,7,26). Due to the altered metabolism in malignant cells, targeting glycolysis may provide a novel approach in the treatment of cancer.

A variety of agents have been explored to selectively suppress glycolysis. Some representative examples are DCA, 2-deoxyglucose, IAA and 3-BrPA. DCA activates the pyruvate dehydrogenase complex by targeting pyruvate dehydrogenase kinase (27). 2-Deoxyglucose can non-competitively inhibit hexokinase II to block glycolysis (28). IAA is reported to inhibit glyceraldehyde-3-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase (29). However, the exact underlying molecular mechanisms of these drugs are still under further investigation. Previous studies from Pedersen and our team had revealed that 3-BrPA decreased the glycolysis rate by suppressing mitochondrial hexokinase activity (9,10). Similarly, SCT has also been revealed to inhibit medullary thyroid cancer, leucocythemia, malignant pleural mesothelioma and ovarian carcinoma growth (1013). On account of their potential ability in the treatment of malignant tumors, we employed 3-BrPA and SCT in the present study and attempted to reveal their underlying antitumor mechanisms. As shown in Fig. 5B and C, 3-BrPA and SCT decreased lactate and ATP production by targeting HK and PFK-1, respectively. The decrease of ATP and lactate may contribute to decrease the energy supply and disrupt the acidic microenvironment, resulting in tumor growth inhibition and apoptosis induction (Figs. 2 and 4).

In addition, based on the high glycolytic rates in malignant cancer, PET imaging with 18F-FDG has been extensively used for clinical detection of tumors (30). Moreover, small animal PET/CT imaging has been frequently utilized to detect, monitor metastasis and evaluate therapeutic response in animal experiments (31,32). Due to the aforementioned rationale, in the present study, micro-PET/CT was employed to detect the accumulation of 18F-FDG in gastric orthotopic xenografts to reflect their metabolic activity and viability. As shown in Fig. 3, the 18F-FDG uptake decreased in the 3-BrPA, SCT and 5-FU-treated groups. The changes in radioactivity uptake were significantly correlated with the inhibition of HK and PFK-1 activities (Fig. 5C). In view of these results we suggest that a decrease in the uptake of 18F-FDG reflects the glycolysis inhibition of tumors.

Apoptosis, known as a programmed cell death, is frequently utilized to evaluate the potential anticancer ability of a new therapeutic approach (33). To understand the effect on the apoptosis of 3-BrPA and SCT, we employed Hoechst 33258 staining. From the results of the Hoechst 33258 staining, we found that apoptosis was induced by 3-BrPA and SCT in a time- and concentration-dependent manner in vitro (Fig. 1A and C). In addition, the results of a TUNEL staining experiment in our previous study also indicated that 3-BrPA and SCT promote tumor cell apoptosis in a dose-dependent manner in vivo (9). Moreover, the typical morphological features of cell apoptosis were observed by TEM (9), and further demonstrated that 3-BrPA and SCT could induce apoptosis with intraperitoneal administration.

It is well known that there are 2 major signaling pathways that trigger apoptosis in mammalian cells: the extrinsic (death receptor) pathway and the intrinsic (mitochondrial) pathway. The extrinsic pathway can immediately activate caspase-8 by death receptors on the cell surface. However, in the intrinsic pathway, the Bcl-2 family modulates the mitochondrial membrane potential to regulate mitochondrial apoptotic signaling (18). Pro-apoptotic Bax promotes permeabilization of the outer mitochondrial membrane, induces the release of apoptogenic proteins such as cytochrome c (Cyt-C) into the cytoplasm and subsequent activation of caspase-9 (1416). Conversely, anti-apoptotic Bcl-2 can negatively regulate the activity of Bax to inhibit apoptosis. Thus, the expression of Bax, Bcl-2 and Cyt-C proteins in tumors was assessed by western blot analyses. Our results indicated that when treated with increasing levels of 3-BrPA and SCT, the expression of the Bax and Cyt-C proteins gradually increased, while the expression of the Bcl-2 protein gradually decreased (Fig. 5A).

Moreover, the Cyt-C release from the mitochondria into the cytosol, forms apoptosomes with Apaf-1 and caspase-9, which ultimately activates caspase-3 (34). Activated caspase-9 could be regarded as an indicator of the mitochondrial-mediated apoptotic pathway and activated caspase-3 is the intersection of the extrinsic and intrinsic pathways. Both play critical roles in the execution of apoptosis (17). In the present study, the expression levels of cleaved caspase-9 and −3 were assessed by IHC staining. As shown in Fig. 4, 3-BrPA and SCT expedited the activation of caspase-9 and −3 in a concentration-dependent manner.

It is believed that the generation of ROS can cause uncontrolled oxidative stress, alter the mitochondrial membrane potential and activate caspase-9 to induce subsequent cell apoptosis (22,35,36). Increasing levels of ROS generated in the SGC-7901 cells were observed after a dose-increase of 3-BrPA and SCT treatment (Fig. 1B and D). Combined with the increased expression of Cyt-C, cleaved caspase-9 and −3 in tumor tissues, we speculated that ROS acted as upstream signaling molecules and played an important role in the induction of apoptosis.

In addition, as a member of the IAP family, survivin is abundantly expressed in the majority of human cancers, and numerous studies have revealed that it acts as a critical inhibitor of apoptosis to promote tumor proliferation and treatment resistance (19,37). Furthermore, survivin can inhibit the activation of caspases by directly binding to caspase-9 and −3 (20,21). As shown in Fig. 5A, a marked decrease of survivin in tumors was observed after treatment, which resulted in the upregulation of cleaved caspase-9 and −3. Thus, one of the antitumor mechanisms of 3-BrPA and SCT may rely on the inhibition of survivin to induce apoptosis.

To the best of our knowledge, this is the first demonstration that intraperitoneal injection of 3-BrPA and SCT leads to glycolysis restraint in gastric orthotopic xenografts and that this method is non-invasive and detectable by 18F-FDG PET/CT. The results encourage the application of 18F-FDG PET/CT to detect glycometabolism changes in tumors after treatment, as a considerable strategy to evaluate therapeutic response in pre-clinical research of malignant tumors. Furthermore, the present study revealed the underlying antitumor mechanisms of 3-BrPA and SCT, by suppressing the activities of glycolytic enzymes to decrease ATP and lactate production; increasing generation of ROS, downregulating the expression of survivin and inducing mitochondrial-mediated apoptosis. In addition, no significant pathological alterations in the liver and kidneys were observed after treatment, indicating the low toxicity of 3-BrPA and SCT. Collectively, our findings highlight the potential of intraperitoneal injection of 3-BrPA and SCT as a safe, novel and attractive strategy in gastric cancer treatment.

Acknowledgements

The present study was supported by the National Natural Science Foundation of China (grant no. 81260366), and The Guangxi Scientific Research and Technology Development Project.

References

1 

Siegel RL, Miller KD and Jemal A: Cancer statistics, 2015. CA Cancer J Clin. 65:5–29. 2015. View Article : Google Scholar : PubMed/NCBI

2 

Torre LA, Bray F, Siegel RL, Ferlay J, Lortet-Tieulent J and Jemal A: Global cancer statistics, 2012. CA Cancer J Clin. 65:87–108. 2015. View Article : Google Scholar : PubMed/NCBI

3 

Heiden MG Vander, Cantley LC and Thompson CB: Understanding the Warburg effect: The metabolic requirements of cell proliferation. Science. 324:1029–1033. 2009. View Article : Google Scholar : PubMed/NCBI

4 

Park MJ, Lee WJ, Lim HK, Park KW, Choi JY and Kim BT: Detecting recurrence of gastric cancer: The value of FDG PET/CT. Abdom Imaging. 34:441–447. 2009. View Article : Google Scholar : PubMed/NCBI

5 

Weber WA and Wieder H: Monitoring chemotherapy and radiotherapy of solid tumors. Eur J Nucl Med Mol Imaging. 33 Suppl 1:S27–S37. 2006. View Article : Google Scholar

6 

Najafov A and Alessi DR: Uncoupling the Warburg effect from cancer. Proc Natl Acad Sci USA. 107:pp. 19135–19136. 2010; View Article : Google Scholar : PubMed/NCBI

7 

Zhao Y, Butler EB and Tan M: Targeting cellular metabolism to improve cancer therapeutics. Cell Death Dis. 4:e5322013. View Article : Google Scholar : PubMed/NCBI

8 

Pedersen PL: Warburg, me and Hexokinase 2: Multiple discoveries of key molecular events underlying one of cancers' most common phenotypes, the ‘Warburg Effect’, i.e., elevated glycolysis in the presence of oxygen. J Bioenerg Biomembr. 39:211–222. 2007. View Article : Google Scholar : PubMed/NCBI

9 

Wang TA, Zhang XD, Guo XY, Xian SL and Lu YF: 3-Bromopyruvate and sodium citrate target glycolysis, suppress survivin, and induce mitochondrial-mediated apoptosis in gastric cancer cells and inhibit gastric orthotopic transplantation tumor growth. Oncol Rep. 35:1287–1296. 2016. View Article : Google Scholar : PubMed/NCBI

10 

Kruspig B, Nilchian A, Orrenius S, Zhivotovsky B and Gogvadze V: Citrate kills tumor cells through activation of apical caspases. Cell Mol Life Sci. 69:4229–4237. 2012. View Article : Google Scholar : PubMed/NCBI

11 

Lincet H, Kafara P, Giffard F, Abeilard-Lemoisson E, Duval M, Louis MH, Poulain L and Icard P: Inhibition of Mcl-1 expression by citrate enhances the effect of Bcl-xL inhibitors on human ovarian carcinoma cells. J Ovarian Res. 6:722013. View Article : Google Scholar : PubMed/NCBI

12 

Halabe Bucay A: Hypothesis proved&citric acid (citrate) does improve cancer: A case of a patient suffering from medullary thyroid cancer. Med Hypotheses. 73:2712009. View Article : Google Scholar

13 

Lu Y, Zhang X, Zhang H, Lan J, Huang G, Varin E, Lincet H, Poulain L and Icard P: Citrate induces apoptotic cell death: A promising way to treat gastric carcinoma? Anticancer Res. 31:797–805. 2011.PubMed/NCBI

14 

Jin Z and El-Deiry WS: Overview of cell death signaling pathways. Cancer Biol Ther. 4:139–163. 2005. View Article : Google Scholar : PubMed/NCBI

15 

Ghobrial IM, Witzig TE and Adjei AA: Targeting apoptosis pathways in cancer therapy. CA Cancer J Clin. 55:178–194. 2005. View Article : Google Scholar : PubMed/NCBI

16 

Fulda S and Debatin KM: Extrinsic versus intrinsic apoptosis pathways in anticancer chemotherapy. Oncogene. 25:4798–4811. 2006. View Article : Google Scholar : PubMed/NCBI

17 

Slee EA, Adrain C and Martin SJ: Executioner caspase-3, −6, and −7 perform distinct, non-redundant roles during the demolition phase of apoptosis. J Biol Chem. 276:7320–7326. 2001. View Article : Google Scholar : PubMed/NCBI

18 

Taylor RC, Cullen SP and Martin SJ: Apoptosis: Controlled demolition at the cellular level. Nat Rev Mol Cell Biol. 9:231–241. 2008. View Article : Google Scholar : PubMed/NCBI

19 

Mita AC, Mita MM, Nawrocki ST and Giles FJ: Survivin: Key regulator of mitosis and apoptosis and novel target for cancer therapeutics. Clin Cancer Res. 14:5000–5005. 2008. View Article : Google Scholar : PubMed/NCBI

20 

Shin S, Sung BJ, Cho YS, Kim HJ, Ha NC, Hwang JI, Chung CW, Jung YK and Oh BH: An anti-apoptotic protein human survivin is a direct inhibitor of caspase-3 and −7. Biochemistry. 40:1117–1123. 2001. View Article : Google Scholar : PubMed/NCBI

21 

Tamm I, Wang Y, Sausville E, Scudiero DA, Vigna N, Oltersdorf T and Reed JC: IAP-family protein survivin inhibits caspase activity and apoptosis induced by Fas (CD95), Bax, caspases, and anticancer drugs. Cancer Res. 58:5315–5320. 1998.PubMed/NCBI

22 

Orrenius S, Gogvadze V and Zhivotovsky B: Mitochondrial oxidative stress: Implications for cell death. Annu Rev Pharmacol Toxicol. 47:143–183. 2007. View Article : Google Scholar : PubMed/NCBI

23 

ille EA Sausv and Burger AM: Contributions of human tumor xenografts to anti cancer drug development. Cancer Res. 66:3351–3354. 2006. View Article : Google Scholar : PubMed/NCBI

24 

Johnson JI, Decker S, Zaharevitz D, Rubinstein LV, Venditti JM, Schepartz S, Kalyandrug S, Christian M, Arbuck S, Hollingshead M, et al: Relationships between drug activity in NCI preclinical in vitro and in vivo models and early clinical trials. Br J Cancer. 84:1424–1431. 2001. View Article : Google Scholar : PubMed/NCBI

25 

Bibby MC: Orthotopic models of cancer for preclinical drug evaluation: Advantages and disadvantages. Eur J Cancer. 40:852–857. 2004. View Article : Google Scholar : PubMed/NCBI

26 

Solaini G, Sgarbi G and Baracca A: Oxidative phosphorylation in cancer cells. Biochim Biophys Acta. 1807:534–542. 2011. View Article : Google Scholar : PubMed/NCBI

27 

Bonnet S, Archer SL, Allalunis-Turner J, Haromy A, Beaulieu C, Thompson R, Lee CT, Lopaschuk GD, Puttagunta L, Bonnet S, et al: A mitochondria-K+ channel axis is suppressed in cancer and its normalization promotes apoptosis and inhibits cancer growth. Cancer Cell. 11:37–51. 2007. View Article : Google Scholar : PubMed/NCBI

28 

Singh D, Banerji AK, Dwarakanath BS, Tripathi RP, Gupta JP, Mathew TL, Ravindranath T and Jain V: Optimizing cancer radiotherapy with 2-deoxy-d-glucose dose escalation studies in patients with glioblastoma multiforme. Strahlenther Onkol. 181:507–514. 2005. View Article : Google Scholar : PubMed/NCBI

29 

Fahim FA, Esmat AY, Mady EA and Ibrahim EK: Antitumor activities of iodoacetate and dimethylsulphoxide against solid Ehrlich carcinoma growth in mice. Biol Res. 36:253–262. 2003. View Article : Google Scholar : PubMed/NCBI

30 

Buerkle A and Weber WA: Imaging of tumor glucose utilization with positron emission tomography. Cancer Metastasis Rev. 27:545–554. 2008. View Article : Google Scholar : PubMed/NCBI

31 

Bradbury MS, Hambardzumyan D, Zanzonico PB, Schwartz J, Cai S, Burnazi EM, Longo V, Larson SM and Holland EC: Dynamic small-animal PET imaging of tumor proliferation with 3′-deoxy-3′-18F-fluorothymidine in a genetically engineered mouse model of high-grade gliomas. J Nucl Med. 49:422–429. 2008. View Article : Google Scholar : PubMed/NCBI

32 

Walter MA, Hildebrandt IJ, Hacke K, Kesner AL, Kelly O, Lawson GW, Phelps ME, Czernin J, Weber WA and Schiestl RH: Small-animal PET/CT for monitoring the development and response to chemotherapy of thymic lymphoma in Trp53-/- mice. J Nucl Med. 51:1285–1292. 2010. View Article : Google Scholar : PubMed/NCBI

33 

Talib WH and Mahasneh AM: Antiproliferative activity of plant extracts used against cancer in traditional medicine. Sci Pharm. 78:33–45. 2010. View Article : Google Scholar : PubMed/NCBI

34 

Launay S, Hermine O, Fontenay M, Kroemer G, Solary E and Garrido C: Vital functions for lethal caspases. Oncogene. 24:5137–5148. 2005. View Article : Google Scholar : PubMed/NCBI

35 

Gupta S, Yel L, Kim D, Kim C, Chiplunkar S and Gollapudi S: Arsenic trioxide induces apoptosis in peripheral blood T lymphocyte subsets by inducing oxidative stress: A role of Bcl-2. Mol Cancer Ther. 2:711–719. 2003.PubMed/NCBI

36 

Fan TJ, Han LH, Cong RS and Liang J: Caspase family proteases and apoptosis. Acta Biochim Biophys Sin. 37:719–727. 2005. View Article : Google Scholar : PubMed/NCBI

37 

Church DN and Talbot DC: Survivin in solid tumors: Rationale for development of inhibitors. Curr Oncol Rep. 14:120–128. 2012. View Article : Google Scholar : PubMed/NCBI

Related Articles

Journal Cover

January-2018
Volume 39 Issue 1

Print ISSN: 1021-335X
Online ISSN:1791-2431

Sign up for eToc alerts

Recommend to Library

Copy and paste a formatted citation
x
Spandidos Publications style
Wang T, Xian S, Guo X, Zhang X and Lu Y: Combined 18F-FDG PET/CT imaging and a gastric orthotopic xenograft model in nude mice are used to evaluate the efficacy of glycolysis-targeted therapy. Oncol Rep 39: 271-279, 2018
APA
Wang, T., Xian, S., Guo, X., Zhang, X., & Lu, Y. (2018). Combined 18F-FDG PET/CT imaging and a gastric orthotopic xenograft model in nude mice are used to evaluate the efficacy of glycolysis-targeted therapy. Oncology Reports, 39, 271-279. https://doi.org/10.3892/or.2017.6060
MLA
Wang, T., Xian, S., Guo, X., Zhang, X., Lu, Y."Combined 18F-FDG PET/CT imaging and a gastric orthotopic xenograft model in nude mice are used to evaluate the efficacy of glycolysis-targeted therapy". Oncology Reports 39.1 (2018): 271-279.
Chicago
Wang, T., Xian, S., Guo, X., Zhang, X., Lu, Y."Combined 18F-FDG PET/CT imaging and a gastric orthotopic xenograft model in nude mice are used to evaluate the efficacy of glycolysis-targeted therapy". Oncology Reports 39, no. 1 (2018): 271-279. https://doi.org/10.3892/or.2017.6060