The Function and Its Molecular Mechanism of Apoptosis Induced by Hyperthermia and Radiotherapy in Human Gastric Cancer Cell Lines
|School||Guangzhou Medical College|
|Keywords||Gastric cancer Hyperthermia Radiotherapy Apoptosis Molecular mechanism|
Gastric cancer is one of the most common malignant tumors in gastrointestinal tract, surgical has occupied the dominant position of treatment for a long time, while the overall survival is still not satisfactory. Radiation therapy is one of the three major treatment in cancer, either use it alone or combined with other therapies, it plays an important role in tumor therapy. The application of hyperthermia combined with radiotherapy is complementary and has synergistic sensitizing effect, which can significantly improve the outcome of gastric cancer. The combined effects of hyperthermia and radiation induced apoptosis is the major way of its synergistic anti-tumor effects in gastric cancer cells. However, the synergistic effect of apoptosis induced by hyperthermia combined with radiotherapy in gastric cancer cell lines based on few studies, the molecular signal transduction mechanisms are not yet fully understood. This study made an initial exploration about the synergistic effect of the heat radiosensitization and its molecular mechanism at the cellular level and molecular level on both the functional and its mechanism, to provide a theoretical basis for the application of the combine therapy.Part One: The Apoptosis Induced by Hyperthermia and Radiotherapy in Human Gastric Cancer Cell LinesObjective:1. To investigate the relations of apoptosis rate - radiation dose, apoptosis rate– time and apoptosis rate - temperature induced by hyperthermia and radiotherapy and its combination therapy on human gastric cancer cells MGC7901 in vitro, explore the trend on each group and identify the best radiation dose, temperature and time fit for apoptosis detection;2. To compare the superiority of the combination therapy with hyperthermia and radiotherapy in the two kinds of cancer cells MGC7901 and MGC803, prove the synergistic action of combination therapy;3. According to the best radiation dose, temperature and time identified in SGC7901, detect the apoptosis rate and the cell cycle in the two kinds of cancer cells MGC7901 and MGC803 with flow cytometry (FCM).Materials and Methods:1. The cell line: select the human gastric cancer cell line of SGC7901 and MGC803, cultivated with conventional cell culture technology.2. Cell radiation and heat treatment methods: (1) Cell Radiation: Setting the source skin distance 100 cm isocentric irradiation method, the flask placed directly on the plates were irradiated with linear accelerator, according to the thickness of the flask as the depth of radiation 2.5 cm, this method could achieve the intended purpose; (2) Cell Hyperthermia: put the cell culture bottle which were need for heat treatment on the level frame into the water bath, water height was above the bottom of the flask about 0.5 cm ~ 1 cm, according to the needs of different groups to water bath ,The temperatures were set at 39, 41, 43 and 45℃, remove the culture bottle into the CO2 incubator at 37℃after 60 min heat treatment;3. Detection of apoptosis: Centrifugate the prepared single cell suspension of each treatment group and discard the supernatant then operate according to the operating instructions of apoptosis detection kit: Adding 500μl of 1×Binding Buffer, add 5μl Annexinⅴ-FITC, then add 5μl PI, incubated for 15 min at room temperature and then can be detected on the machine;4. Detection of cell cycle: The cell concentration of the single cell suspension was adjusted to about 1.0×106 cells / ml, room temperature and centrifugated cells can be dyed after the supernatant discarding: (1) add 250ul A solution to each tube, gently mix, not to vibrate it, room temperature for 10 min; (2) by adding 200ul B solution to each tube, gently mix, not to vibrate it, room temperature for 10 min;(3) each tube add 200ul C solution, mix gently, do not vibrate it, low temperature (2 ~ 8℃) dark standing 10 min; (4) filter cells with omentum or 35um 50 um nylon cell filter, low temperature and avoid light place to be ready for detection; 5. Judgement of results: On the two-variable flow cytometry scatter plots, the lower left quadrant shows living cells, upper left quadrant, necrotic cells, early apoptotic cells in the lower right quadrant, late apoptotic cells in the upper right quadrant. The lower right quadrant and the upper right quadrant of the cells was recorded as the total rate of apoptosis;6. Statistical methods: Using SPSS 17.0 statistical analysis software, completely randomized design one-way analysis of variance and pairwise comparison of apoptosis rates in each treatment group, when P <0.05 the difference was statistically significant.Results:1, cell morphological changesReceived different degree of heat therapy, radiation and heat radiation therapy joint function, the MGC803 and SGC7901 cells in each group occurr different degree of apoptosis, the common features of its apoptosis are as follws: the adjacent cells ,which grow with wall, lose their connections, shrinkages and rounded, outlines show more clearly visible, cells ability is abate and they can easily fall from the wall of the training bottles;2, flow cytometric art experimental results2.1, Radiotherapy dose-dependent experiments, SGC7901 cells in the control group and 24 h after 2, 5 and 10 Gy radiotherapy, the apoptosis rate of 3 results was expressed as x±s: 2.33%±0.89%, 6.32%±0.88%, 28.16 %±1.45% and 9.95%±2.46%, the difference between the groups was statistically significant (P <0.05 );2.2, Apoptosis rate of radiotherapy - time variation, SGC7901 cells in the control group and 12, 24 and 48 h after 5 Gy radiotherapy, the apoptosis rate of 3 results was expressed as x±s: 2.99%±0.68%, 6.14%±0.86%, 28.16%±1.45% and 8.77%±2.39%, the difference between each group was of statistical significant (P <0.05) except the 12 h group with the 48 h group was of no statistical significant(P =0.064);2.3, Temperature - dependent experiments, SGC7901 cells in the control group and 24 h after 39, 41, 43 and 45℃hyperthermia; the apoptosis rate of 3 results was expressed as x±s: 2.99%±0.68%, 7.45%±1.65%, 32.14%±2.92%, 44.42%± 5.58% and 33.50%±2.87%, the difference between each group was of statistical significant (P <0.05) except the control group with the 39℃group (P=0.118) and the 41℃group with the 45℃(P=0.685) was of no statistical significant;2.4, Selected 24 h and 43℃as the testing time and processing temperature of FCM analysis, analyse the the apoptosis rate of the two cells in the control group and after 2 Gy, 5 Gy, 10 Gy, 43℃, 2 Gy + 43℃, 5 Gy + 43℃, 10 Gy + 43℃: The apoptosis rate of 3 results in SGC7901 was expressed as x±s: 3.25%±0.72%, 10.89%±2.69%, 22.21%±2.05%, 14.21%±0.91%, 28.73 %±2.76%, 47%±4.86%, 33.92%±4.36% and 22.26%±2.37%, while the MGC803 cell was: 3.78%±1.55%, 9.39 %±1.8%, 22.35%±1.64%, 15.37%±1.89%, 40.59%±2.27%, 53.82%±1.13%, 36.67%±1.18% and 32.91%±1.96%. The difference between each group was of statistical significant (P <0.05) except the 2 Gy group with the 10 Gy group (P=0.199) and the 5 Gy group with the 10 Gy radiation combined with 43℃hyperthermia group (P=0.986) in SGC7901 was of no statistical significant.What worthes concern is that, the apoptosis rate of the 2 Gy + 43℃group was higher than the sum apoptosis rate of the 2 Gy radiotherapy and the 43℃hyperthermia alone, and the difference between them was of statistical significant (P <0.05);2.5, Selected 24 h and 43℃as the testing time and processing temperature of FCM analysis, analyse the cells cycle of the two cells in the control group and after 2 Gy, 5 Gy, 10 Gy, 43℃, 2 Gy + 43℃, 5 Gy + 43℃, 10 Gy + 43℃: The percentage of G0/G1 phase cells and the percentage of S phase cells were shown the opposite changes in the two human gastric cancer cell lines. The percentage of G0/G1 phase cells in SGC7901 were 58.17%, 51.56%, 40.97%, 46.95%, 23.55%, 23.69%, 16.48% and 20.36%, the percentage of S phase cells were 33.83%, 40.44%, 51.03%,45.05%, 51.6%, 51.34%, 75.52% and 71.64%, while the percentage of G0/G1 phase cells in MGC803 were 23.69%, 24.99%, 45.87%, 62.08%, 23.3%, 23.68%, 12.94% and 18.73%, the percentage of S phase cells were 37.18%, 67.01%, 46.13%, 29.92%, 52.16%, 51.46%, 79.06% and 74.27%. Conclusions:1, human gastric cancer cells SGC7901 and MGC803 have different levels of apoptosis to the varying degrees of hyperthermia, radiotherapy and its combine effects at different time, the morphologic features are closely related to the severity of the treatment factors; the morphological changes of the poorly differentiated MGC803 cells are more obvious than the SGC7901 cells;2. 5 Gy, 43℃and 24 h is the appropriate apoptosis processing factors and the detection time for SGC7901 cells, 2 Gy + 43℃is the appropriate apoptosis processing factors of combine treatment; the combined effects has higher rates of apoptosis than radiation or hyperthermia alone and showed a significant thermal radiation synergy. The poorly differentiated MGC803 cells has a higher rate of apoptosis than the moderately differentiated SGC7901 cells to the same treatment. 3. with the apoptosis occur, the cell cycle also changes regularly, the percentage of G0/G1 phase cells descended and S phase cells ascend, both cells mainly occurred the S phase of the cell cycle arrest;Part Two: The Molecular Mechanism of The Apoptosis Induced by Hyperthermia and Radiotherapy in Human Gastric Cancer Cell LinesObjective:1. Applicate the Western blot technique to detect the expression and content of apoptosis-related protein in the two human gastric cancer cells, find the hyperthermia combined with radiotherapy have advantages on the expression of apoptosis-related protein than the hyperthermia and radiotherapy alone, and indirectly confirmed the hyperthermia combined with radiotherapy have synergy;2. Compare the expression levels of apoptosis-related protein in the two human gastric cancer cells, infer the role of the combine therapy of hyperthermia and radiotherapy in apoptosis signal transduction pathways, intially clarify the molecular mechanism of hyperthermia combined with radiotherapy. Materials and Methods:1. The Western blot solution preparation: the relevant solution in this experiment was preparaed according to the statement, reagent manual or "Molecular Cloning Laboratory Manual (Third Edition)";2. Detection of the protein concentration: adopt the enhanced BCA protein concentration assay kit of the beyotime company, calculate the protein concentration of samples in each group and the sample volume through measuring the absorbance of the standard protein and the absorbance of the samples. Operation steps: (1) take 0.8 ml of the standard preparation of protein solution to a protein standard (20 mg BSA), fully dissolved into the preparation of 25 mg/ml of protein standard solution; (2) take appropriate 25 mg/ml protein standard, diluted to a final concentration of 2 mg/ml; (3) preparation of the BCA working solution (50:1), mix well; (4) added the standard solution of 0, 1, 2, 4, 8, 12, 16, 20μl to the standard 96 holes, plus standard dilution complement to 20μl; (5) Add the appropriate volume of sample to standard 96 holes, plus the standard dilution solution to 20μl; (6) add 200μl BCA working solution to each hole, 37℃temperature for 20 to 30 minutes; (7) detect the A562, calculate the protein concentration of samples according to the standard curve;3. Western blot technique: (1) sample processing: pyrolysis treated cells, measure the concentration, take the same amount of protein sample added directly after the adding of loading buffer; (2) Electrophoresis: After all the protein sample added, marker also added with 1×loading buffer adjusted to equal volume with the sample, add marker 5μl, each protein sample 30μl, stop when the target protein swimming to 1 cm above the lower edge of the end; (3) transfer film: The PVDF membrane soaked in methanol, 5 min, transferred to electroporation solution, remove the glue, keep target band region, immersed it in the transfer of fluid, with each side of the membrane covered by the order of a filter paper, avoid air bubbles; (4) Electrical transfer: add 1 000 ml electroporation solution into the electricity transfer slot, the glue and the PVDF membrane tile in the filter paper placed as sandwich and lied on the sponge, tightly closed again after the expulsion of air bubbles into the electricity transfer slot, keep electrodes in the right direction; 100V power 1 h for electrical transfer at low temperature; (5) closure and hybridization: remove the membrane from the transfer slot, PBST rinsing three times, 5 ~ 10 min for each time, discard PBST then immersed in confining liquid and sway slowly for 1 h, one hour later rinsing with PBST as former and then add the diluted antibody at 4℃for overnight; PBST rinse as former after incubation; Secondary antibody inucubation for one hour at room temperature and sway slowly; (6) ECL chemiluminescence reaction: Take 350μl of ECL reaction A and B mixed them sufficiently, dry the membrane with filter paper and place it on the glass of the molecular imaging system, set system parameters and the exposure time as 5, 10, 15 min, preservation and analysis;4. Choice of antibody and related reagents: Focused on the mitochondrial apoptosis pathway, death receptor pathway and the pathway of the endoplasmic reticulum, we select the antibody of apoptosis-related protein phosphorylated P53 (p-P53), cleaved Caspase 9, Caspase 3, Caspase 8 and Caspase 12 for western blot experiments.Results:The expression of apoptosis-related proteins p-P53, c-Caspase 9, c-Caspase 8 and c-Caspase 3 were detected the highest levels in the hyperthermia alone group was at the 43℃sub-group, and the highest levels in radiation alone group was at the 5 Gy sub-group, the highest levels in the radiation combined hyperthermia group was at the 2 Gy + 43℃sub-group. The result ofβ-actin demonstrated the homogeneity of sample volume in each groups is well.Conclusions:1. The highest expression levels of apoptosis-related proteins of two human gastric cancer cells SGC7901 and MGC803 in radiotherapy, hyperthermia and combine treatment groups were respectively at 5 Gy, 43℃and 2 Gy + 43℃sub-group and the combined treatment group was the highest. This confirmed synergy effect of the hyperthermia and the radiotherapy from the molecular level;2. Mitochondrial pathway and death receptor pathway play important roles in the apoptosis induced by the combined treatment of hyperthermia and radiation in human gastric cancer cells SGC7901 and MGC803, while the endoplasmic reticulum pathway may have some degree of effect.