| Background:Increased glycolysis in cancer, a phenomenon known as the Warburg effect, has been observed in various tumor cells and represents a major biochemical alteration associated with malignant transformation. However, many cancer cells still possess various degrees of mitochondrial respiration activities to various extents, and different cancer cells may have different pathways to acquire energy, not relying on glycolysis as their sole pathway to generate ATP. A clear understanding of the different dependence on energy metabolism in cancer cells and its complex metabolic regulatory mechanisms is essential for the successful development of new therapeutic agents and effective regimens for cancer treatment.Previously, energy metabolic materials of cultured cells and tissues were investigated by enzymatic analysis method, which was difficult to be promoted because it was low sensitivite, slow and more complex to be operated. Then, reversed phase high performance liquid chromatogrophy (RP-HPLC) wae used (phosphate dihydrogen potassiumbuffer as the mobile phase and gradient elution). It is sensitive, however, if it will be used for a long time, the column will be seriously damaged because of its mobile phase and gradient elution. Thus, establishment of a steady and sensitive RP-HPLC method is necessary for detection of energy metabolic materials of cells under different conditions and of animal tissues.Objectives:1. Establishment of a steady and sensitive RP-HPLC method for detection of energy metabolic materials of cells under different conditions and of animal tissues.2. Determination of the main energy-dependent manners in several cell lines, aiming to provide basis for the development of new anti-tumor therapeutic strategies.3. Determination of the energy metabolic materials in different animal tissues.4. Investigation of the protective function of the external energy against the UUO (Unilateral ureteral obstruction)-induced renal fibrosis and CCl4-induced liver injury, which in order to provide some laboratory indicators for other prevention and treatment. Methods:1. Establishment of an optimized RP-HPLC method:(1) Optimization of HPLC for determination of ATP, ADP and AMP:According to the physical, chemical properties of energy metabolic materials(ATP, ADP and AMP), the appropriate column, detector's wavelength, chromatographic analysis, mobile phase, the pH value of the mobile phase, flow rate and column temperature were selected.(2) Establishment of the methodology of energy metabolic material:A. Standard curve: under the selected analysis condition, by external standard quantitative method, the ATP, ADP and AMP concentrations used for establishment of the standard curve of organization and cell being 0, 5, 10, 25, 50, 100 and 200μg/mL for tissues; 0, 0.05, 0.1, 1, 5, 25, 50, 100 and 200μg/mL for cells.B. Precision: three concentrations (low, medium and high) used in intra-day and inter-day precision tests, tested for 5 times each day in intra-day precision test, and for once a day for 5 days continuously in inter-day precision test.C. Recovery rate: three different concentrations (low, medium and high) of standard hybrid of ATP, ADP and AMP added to the samples of known concentration respectively, according to the sample test sample and their recovery rates determinated.D. Qualitative separation of ATP, ADP and AMP: three samples (standard, sample and sample plus standard) tested respectively, and their peak times, peak areas and compositions determined.2. Establishment of the cell models under different metabolic conditions:(1) Selection of 10 kinds of cell lines. Cell lines are ECV304 (human umbilical vein endothelial cells) (DMEM cultured including 1g/L glucose), A549 (human lung adenocarcinoma cell) (RPMI-1640 cultured), K562 (human chronic myelogenous leukemia cells) (RPMI-1640 cultured), H460 (human lung cancer cells) (RPMI-1640 cultured), MCF7 (human breast cancer cells) (RPMI-1640 cultured), P388 (mouse leukemia cells) (RPMI-1640 cultured), C6 (rat glioma) (DMEM cultured), Ana-1 (murine macrophage) (RPMI-1640 cultured), SH-SY5Y (human neuroblastoma) (DMEM cultured) and thymus cells(RPMI-1640 cultured).(2) Establishment of cell models in different metabolic conditions: Optimized RP-HPLC method applicated in investigating the main energy-dependent manners in several cell lines. Under conditions, with glucose or without glucose, in hypoxia or normoxia, with L-glucose or D-glucose, concentration gradient of glucose, adenosine and oligomycin concentration gradient, as well as drugs such as adenosine, oligomycin, LPS and 2-deoxyglucose.3. Measurement of their energy metabolic matrials in tissues and establishment of CCl4-induced liver injury model and UUO-induced renal fibrosis model .(1) Comparison of the energy metabolic materials in different organs of normal mice: three healthy Kunming male mice, 18 ~ 20g. The model of heart, lung, kidney, brain and skeleton of mice were taken out, the energy metabolic contents of their various organs were detected by optimized RP-HPLC method.(2) Establishment of an acute CCl4-induced liver injury model: a total of 20 Kunming mice were each about 18-20g, randomly divided into 4 groups: control group, CCl4 group (0.3mL/100g), FDP (fructose-1,6-bisphosphate) group (2g/kg) and CCl4 plus FDP group, with intraperitoneal injection of corresponding drugs. After 4 hours, FDP were intraperitoneally injected again. And after another 4 hours, livers were removed and the energy metabolites were detected by optimized RP-HPLC.(3) Establishment of UUO-induced renal fibrosis model: a total of 60 Kunming mice were each about 18-20g, randomly divided into 4 groups: sham group, surgery control group, glucose group (2g/kg) and FDP group (2g/kg). Surgical control, glucose and FDP groups were ligated twice in the lower 1/3 of the left ureter; the left ureter of Sham group was not ligated. Mice were intraperitoneally injected corresponding drugs for once a day, once three days or once seven days. Then the kidneys were removed and the energy metabolites were detected by optimized RP-HPLC.Results1. Establishment of an optimized RP-HPLC method:HPLC C18 water resistant column (5 um, 250mm×4.6mm) of license YMC, 254nm wavelength of SPD-10Avp detector, RP-HPLC analysis, 180mM of phosphate dihydrogen potassium (containing 5% methanol and pH value = 6.25) buffer as the mobile phase, 0.8mL/min as flow rate and below or equal to 25℃as the column temperature were selected.2. Establishment of the methodology for detection of energy metabolic materials: (1) Establishment of the standard curve of energy metabolic materials of tissues and cells: It was shown that the concentration and peak area had a good linear relationship.(2) Precision and recovery: It was shown that intra-day and inter-day precisions had a good repeatability, with both the relative standard deviation (RSD) below 1.5% and 5.1%; the recovery rate for ATP was 99% ~ 108%, ADP 96% ~ 104%, AMP 94% ~ l19%.(3) Qualitative separation of ATP, ADP and AMP: the retention time of sample's peak was similar to standard. When adding a mixed standard, three peaks of samples were increased and had a dose-effert relationship to the standard. Thus, the components of the samples'adenylate chromatogram were ATP, ADP and AMP.3. Energy metabolic materials of cell models under different metabolic conditions:(1) Under conditions of glucose or glucose-free culture, the cells'ATP were measured. It was shown that when cells'density was more than 1×106 or/and time of being cultured was longer than 6h, the glucose'ATP was 2 times as glucose-free culture one's (p<0.05).(2) SH-SY5Y cells were cultured for 6 hours with oligomycin (1μg/mL). In conditions of glucose and glucose-free culture, the ATP content of SH-SY5Y cells was decreased obviously (p<0.01). SH-SY5Y cells were cultured for 3 hours with 2-DG (25mM). In conditions of glucose, the ATP content was decreased slightly. While in the absence of glucose, the ATP content was decreased significantly, being about half of the control group (p<0.01). SH-SY5Y cells were cultured for 6 hours in conditions of glucose or glucose-free and normoxic or hypoxic culture, It was shown these cells'ATP content increased slightly (p<0.05).(3) H460 cells and C6 cells were respectively cultured for 6 hours with oligomycin (1μg/mL). In conditions of glucose and glucose-free culture, the ATP content was decreased obviously (p<0.01). In addition, adenosine (ade) can significantly increase the ATP content (p<0.01); If ade and oligomycin being added at the same time, the ATP content treated only with oligomycin could be recovered partly (p<0.01).(4) A549 cells were cultured for 3 hours in conditions of glucose and normoxic or hypoxic culture with oligomycin (1μg/mL) and adenosine (2mM). It was shown there was no significant difference between the conditions of normoxic and hypoxic culture in the ATP content. In conditions of glucose, oligomycin could not reduce ATP content obviously. However, adenosine could significantly increase it (p<0.01); In addition, the ATP content in conditions of glucose-free and normoxic culture was a little more than the conditions of glucose-free and hypoxic culture (p<0.05). Oligomycin could reduce ATP content obviously in conditions of glucose culture (p<0.01). In the same condition, adenosine could increase ATP content evidently (p<0.01).(5) MCF7 cells were cultured for 6 hours with oligomycin (1μg/mL). In conditions of glucose culture, the ATP content wasn't decreased obviously, which was oppositing to the conditions of glucose-free culture. In addition, adenosine could significantly increase its ATP content (p<0.01); If adding ade and oligomycin at the same time, the ATP content treated only with oligomycin could be recovered completely (p<0.01).(6) K562 cells were cultured for 24 hours with oligomycin (0, 0.5, 1, 2, 5 and 10μg/mL). It was shown that in condition of glucose, the ATP content was not changed. But in condition of glucose-free, the ATP content was obviously reduced (p<0.01). K562 cells were cultured for 12 hours under the condition of glucose concentration gradient. It was shown that the concentration of ATP content had also increased along with the increasd and concentration of 0g/L, 1g/L and 2g/L (p<0.05). However, under the concentrations of 4g/L, 6g/L and 8g/L, the ATP concentration did not change. K562 cells were cultured for 6 hours in conditions of glucose and glucose-free culture, with different concentrations adenosine (0, 0.5, 1, 2, 4 and 8mM). It was shown that the ATP content increased as the concentration of adenosine increasd (p<0.05). But the ATP content of concentration of adenosine at 4mM equaled to concentration of adenosine at 8mM. K562 cells was cultured for 6 hours under the conditions of L-glucose (2g/L) or D-glucose (2g/L), it was shown that the ATP content did not change in condition of L-glucose cultured, but was significantly increased in the condition of D-glucose cultured (p<0.05). In condition of glucose, K562 cells were cultured for 3 and 12 hours respectively with 2-DG (5mM) and oligomycin ( 0.5μg/mL). It was shown that after 3 hours 2-DG (5mM) could not obviously reduce the ATP content , but after 12 hours, the ATP was reduced by half (p<0.01).(7) P388 cells were cultured for 6 hours with oligomycin (0, 0.5, 1, 2, 5 and 10μg/mL). It was shown that in condition of glucose, the ATP content was not changed. But in condition of glucose-free, the ATP content was obviously reduced (p<0.01).(8) ECV304 cells were cultured for 3h with oligomycin (2.5μg/mL) and adenosine (2mM). It was shown that in condition of glucose, the ATP content was not changed by oligomycin. But in condition of glucose-free, the ATP content was obviously reduced (p<0.01) by oligomycin. Then ade could increase the ATP content obviously no matterm whether the culture condition was glucose or glucose-free (p<0.01). Adenosine and oligomycin being added at the same time, the ATP content treated only with oligomycin could be recovered completely (p<0.01). ECV304 cells were cultured for 3 hours in the condition of glucose with 2-DG (25mM), the ATP content was not changed. And cultured for 3 hours in the absence of glucose, the ATP content was decreased significantly, being about half of the control group (p<0.01). ECV304 cells were cultured for 3 hours with FDP (25mM), the ATP content was increased slightly (p<0.05) .(9) In conditions of glucose and with oligomycin (4μg/mL), thymus cells were cultured for 10 hours with different concentrations of adenosine (0, 0.1, 0.2, 1 and 2mM). It was shown when concentration of adenosine in 0, 0.1 and 0.2mM, the ATP content was not changed; when the concentration of adenosine concentration was 1mM and 2mM, the ATP content increased (p<0.05) . However, in conditions of glucose and with adenosine (2mM), thymus cells were cultured for 10 hours with different concentrations of oligomycin (0, 1, 2, 4 and 8μg/mL). It was shown when concentration of oligomycin was 0μg/mL, the ATP content was about 2.5μg/mL; when the concentration of oligomycin was 1, 2,4 and 8μg/mL ,the ATP content decreased to about 0.5μg/mL (p<0.01) . In conditions of glucose and glucose-free, thymus cells were cultured for 4 hours with different concentrations of adenosine (0, 0.5, 1, 2 and 4mM). It was shown that in the concentration of 0-2mM stage, the ATP content increased with the drug concentrations increasd (p<0.05) . In the concentration of 4mM, the concentration of ATP concentration was less than 2mM (p<0.05).(10) Ana-1 cells were cultured for 6 hours with oligomycin (2μg/mL) and Ade (2mM). It was shown that in condition of glucose, oligomycin reduced the ATP content by half (p<0.01). Comparing to the control group, adenosine increases the ATP content by half (p<0.01). But in conditions of glucose-free, oligomycin reduces the ATP content obviously (p<0.01). Comparing to the control group, adenosine increased the ATP content by half (p<0.01). In conditions of glucose and glucose-free, Ana-1 cells were cultured for 2 hours and 6 hours with adenosine (2mM) and lipopolysaccharide (LPS) (1μg/mL) respectively. It was shown that LPS could reduce the ATP content in various degrees, while with adding adenosine in the same time, the ATP content was much more than with LPS alone (p<0.05) . 4. Energy metabolic materials in various tissues of mouse and mouse models of fibrosis:(1) Comparison of the energy metabolic contents in the organs of normal mice. It was shown that the highest ATP content was in skeletal muscle, followed by muscles of the heart, the lungs, brain and kidney (p<0.05).(2) Energy metabolic materials in CCl4-induced liver injury model. It was shown ed that the ATP content in CCl4 group is much less than in the control group (p<0.05), and the ATP content in CCl4 plus FDP group twice as high as in CCl4 group (p<0.05).(3) Energy metabolic contents in UUO-induced renal fibrosis. It was shown that the ATP contents in the control, glucose and FDP groups were all increased gradually (p<0.05).Conclusions:1. RP-HPLC methodology to detect energy metabolic materials is proved to be steady and sensitive.2. Various cell lines possess different main energy-dependent manners.3. Adenosine can be used as an external energy replenisher, which can increase the ATP content of cell lines used in the study.4. Different animal tissues in normal mouse possess different levels of energy metabolic materials.5. FDP can increase energy metabolic materials in vivo, which used as an external energy for CCl4-induced liver injury model and UUO-induced renal fibrosis model.
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