| Gastric cancer is the third leading cause of cancer death and was the second common cancer in China. Patient’s quality of life and prognosis were poor due to the progressed stage of the disease before diagnosed. Although surgery and chemotherapy can be curative for early-stage disease, but the prognosis for patients with advanced disease is poor. Targeted prevention and treatment of peritoneal metastasis of gastric carcinomato improve the quality of life in patients with gastric cancer, improve the prognosis of patients has important significance. In recent years, the hyperthermic intraperitoneal chemotherapy (HIPEC) in postoperative gastric cancer recurrence with malignant ascites and abdominal small metastases has made certain achievements,but the exact mechanism is still a lack of in-depth study.Due to the heat resistance of tumor cells than the normal cells, so the HIPEC could remove the drug sensitivity of tumor cells more effectively enhanced by high temperature and induction of autophagyin perfusion, which has an important role in the apoptosis of tumor. Autophagy is related with tumor occurrence and development, which is a hot spot in international cancer research in recent years. Autophagy could promote the tumor cell death under different conditions. However, recent studies suggested that under some conditions, autophagy may be beneficial to promote tumor cell survival. Tumor cells can tolerate a stronger external injury and repair the tumor cell damage, such as resistant to radiation therapy and chemotherapy blow through raising the base level of autophagy. However, excessive inducd autophagy could increase autophagic cell death in tumor cells autophagic cell death, which achieving the purpose of treatment on tumor. At present, Rapamycin, a activator of autophagy has been used to treat a wide variety of tumors, and has a certain effect. Therefore, autophagic cell death may be one of the important strategy of tumor treatment in recent years.It has been reported that reactive oxygen species (ROS) could cause autophagic cell death through a variety of ways, such as the mitochondrial pathway.And ROS induced the dissociation of autophagy-related proteins Beclin 1 and Bcl-2, leading to the activation of Beclin 1-inducd autophagy pathway. Meanwhile, autophagy-related proteins ATG4/LC3II and ATG5 also could induce autophagy and showed that the express on level of autophagy-related molecules, such as Beclin 1, LC3 was raised, and tumor cells could not repair functions normally and result in death.The new study found:thermo-chemotherapy can promote the generation of oxidative stress in tumor cells, improve the level of ROS. thermo-chemotherapy combined with oxidative stress inducer t-BOOH, can increase the intracellular level of ROS, enhance the killing effect on tumor cells.The high expression of ROS thermo-chemotherapy stimulation of tumor cells, inducing tumor cell apoptosis, may be an important mechanism of anti tumor thermo-chemotherapy.According to the above analysis, our study puts forward the following hypothesis:hyperthermia chemotherapy may promote the tumor cells to produce oxidative stress reaction, and increase ROS level of tumor cells, which induce autophagic cell death of gastric cancer cells, which may be one of the important mechanism of the thermo-chemotherapy. To test this hypothesis, the project is studied in two aspects of cytology test and animal model, first and then simulate the thermo-chemotherapy conditions,research produced by ROS expression and correlation of autophagy gene in gastric cancer cells, to investigate the thermo-chemotherapy to produce ROS in autophagic cell death of role in stimulating gastric cancer cells; further study ROS induced autophagy in gastric cancer cell deaththermo-chemotherapy in gastric cancer cell killing effect. Research will provide new ideas for the mechanism of intraperitoneal HIPEC on the prevention and treatment of implantation metastasis of gastric cancer peritoneal.Objective1. Study the relationship of ROS, induced by hyperthermia chemotherapy treatment, and the expression of autophagy-related genes in gastric cancer.2. Explore the effect of ROS, induced by hyperthermia chemotherapy treatment, on autophagic death of gastric carcinoma cell.3. Study ROS, induced by hyperthermia chemotherapy treatment, induced autophagic death behavior of gastric carcinoma cell by producing.Materials and Methods1. Evaluated the therapeutic efficacy on hyperthermia chemotherapy treatment in cell level1.1 The concentration of the drug screening of oxaliplatinThe human gastric carcinoma cell line SGC-7901 was treated with oxaliplatin:When the SGC-7901 enter in the exponential phase, the oxaliplatin was added into medium on different concentration (10μg/ml、20μg/ml、40μg/ml、80μg/ml、160μg/ml and 320μg/ml) for different assay. After oxaliplatin treatment 24 h, cell viability was determined by MTS assay to determine the into the best work concentration of oxaliplatin. IC50 was obtained from three independent experiments, and the oxaliplatin concentration of IC50 were used in the following studies.1.2 Cell treat with oxaliplatin and hyperthermia (HT)Using the best work concentration of oxaliplatin to treat SGC-7901 cell at 1 h, then the cells were treat with different temperature (39℃、41℃、42℃、43℃、45℃) at 1 h for different assays. After incubated for 24 h, cell viability was monitored using the MTS assay to determine the best work temperature.1.3 Group and TreatmentTo ensure the best work concentration of oxaliplatin and the suitable temperature, SGC-7901 cells were randomly divided into four groups, Group 1:control group, Group 2:HT group (treated with HT at the suitable temperature), Group 3: chemotherapy group (treated with oxaliplatin using the best work concentration) and Group 4:Chemotherapy plus HT group (treated with oxaliplatin plus HT at the best work concentration of oxaliplatin and the suitable temperature). The cell cultured at 37℃ was as negative control group.The cells were treated following the above condition for 1 h, continued to culture for 24 h. Then the cells were harvested and the assays were detected as follow:1.3.1 The ROS level of each group was detected by flow cytometry1.3.2 The mitochondrial membrane potential (MMP) was analyzed by flow cytometry1.3.3 The cell viability was monitored using the MTS assay1.3.4 The cell apoptosis was detected by flow cytometry1.3.5 The expression level of autophagy-related proteins Beclin 1, LC3B and mTOR was examined by western blot1.3.6 The change of cellular morphology was observed by optical microscope, the autophagosome was observed by transmission electron microscopy2. Evaluated the therapeutic efficacy on hyperthermia chemotherapy treatment in the aminal level2.1 To establish gastric cancer model using BALB/C-NU nude mouseTo establish gastric cancer model, BALB/C-NU nude mouse (special pathogen-free grade,4-6 weeks old,230-280 g) were used in the study. SGC-7901 cells were subcutaneously injected into the each side of the posterior flank groin region of BALB/C-NU nude mouse. Mice were maintained in a SPF house with 12 h alternating light and dark cycles, and were given adequate nutrition and water. The nude mouse were randomly divided into four groups (n=3):Group 1:control group (in 37℃, injected glucose with the same L-OHP dose into intraperitoneal), Group 2: HT group(in 43℃, injected glucose with the same L-OHP dose into intraperitoneal), Group 3:chemotherapy group (local injection the tumor at a dose of 6 mg/kg oxaliplatin into intraperitoneal) and Group 4:Chemotherapy+HT group (injected at a dose of 6 mg/kg oxaliplatin into intraperitoneal, after chemotherapy performed 1 h, the model were fixed on a special water box. The water need to preheat to 43℃ and the depth of the water in the box can keep tumor region immersed. Put the box into biochemical incubator at 43℃ 1.5 h, to ensure the tumor cell to heat at least 1 h.)2.2 Detect indicators:The animal models cervical dislocation executed after treated with the conditions according to each group at 24 h. The tumors tissue was collected from surgically removed. A part of the tumor tissue incubated in collagenase IV to isolated out single tumor cells. The level of ROS in the cells checked by flow cytometry; Another part of tumor tissue fixed with 10% neutral-buffered formalin, and they were cut into 5-μm sections after embedding in paraffin. The autophagy-related protein of Beclin 1, LC3B and mTOR level were examined by immunohistochemistry. The cell apoptosis of the tumor tissue were detected by TUNEL.2.3 Statistical analysis was performed using origin6.0 and SPSS 19.0 software package (SPSS Inc, Chicago, IL, USA). Continuous Data were expressed as means±sD, The One way ANOVA was used to examine the statistical significant difference between groups, LSD used for the multiple comparisons, factorial design ANOVA used for factorial design data. A p-value of <0.05 (two-tailed) was used to establish statistical significance.Results1. Hyperthermia chemotherapy treatment had synergistic effect in inhibited cell proliferation respect1.1 The L-OHP treatment inhibited SGC-7901 cell viability, and there was a positive correlation between L-OHP concentration and cell viability inhibitory rate.The result of MTS shown that the different concentration of L-OHP could inhibit SGC-7901 cell viability. Upregulating the L-OHP concentration, the SGC-7901 cell viability was signficantly downregulated, and there was a positive correlation between L-OHP concentration and cell viability inhibitory rate. The statistical result shown that the cell viability in eight group had remarkably different (F=1598.325, P=0.000). the cell viability had remarkably different comparison between any two group (P<0.05).According to the result of MTS, the proliferation rate and inhibitory rate were obtained from different concentration of L-OHP. Further, the results indicated that the IC50 was 80.66μg/ml and the L-OHP concentration of 80 μg/ml were use in the subsequent experiments.1.2 The hyperthermia treatment inhibited SGC-7901 cell viability, and there was a positive correlation between temperature treatment and cell viability inhibitory rate.1.1 To research the effect of temperature on the SGC-7901 cell viability, the SGC-7901 cells were treated with different temperature (37℃、39℃、41℃、42℃ 43℃、45℃) when the SGC-7901 enter in the exponential phase. After treated with 80 μg/ml L-OHP 24 h, the SGC-7901 cell were heated at 1 h. The SGC-7901 cells were continued to culture 24 h. Cell viability was monitored using the MTS. The results shown that the cell viability was gradually decreased follewing the increase of temperature, and the result also shown that high-temperature had be able to kill tumor cells. And the statistical result also shown that the interaction between oxaliplatin and temperature was significant in the suppress the cell proliferation, and the main effect of oxaliplatin and temperature were significant in the suppress the cell proliferation (F=25201.956, P=0.000; F=993.630, P=0.000). Further, we used the q value method to analyze whether the L-OHP and HT had cooperation to inhibit the cell viability. The result shown that the L-OHP and hyperthermia started to cooperation when the temperature reached 41℃. Considered the temperature tolerance of the patient in clinical application, we ensured the treated temperature was 43℃.1.3 The L-OHP and hyperthermia inhibit SGC-7901 cell viability had synergistic effect.According to the result of the IC50 and the best treated temperature, we choice the 80μg/ml L-OHP and 43℃ to incubate SGC-7901 cell to demonstrate the synergistic effect. The cell viability was monitored using the MTS. Compared with the control group, the cell viability of HT group, chemotherapy group and chemotherapy+HT group were obviously declined (P<0.05). The reduction of cell viability in chemotherapy+HT group was the highest (P<0.01). the statistical result also shown that the interaction between oxaliplatin and temperature was significant in the suppress the cell proliferation (F=80.948,P=0.000), and the main effect of oxaliplatin and temperature were significant in the suppress the cell proliferation (F=5827.421, P=0.000; F=968.003, P=0.000). Evaluation of Combination therapy effect of L-OHP and HT for SGC-7901 cell viability by q value method and calculate the q value was 1.370, the result shown that the Combination therapy effect of L-OHP and HT for inhibit cell viability had synergism. Moreover, the cells form were observed by inverted microscope. Compared with control group, the cell density of HT group and chemotherapy group were reduced and the cell form became round, and the cell growth of chemotherapy+ HT group were slowest, the cell form became round, the cell floated in the medium, and the cell apoptosis were maximum.2. Combination therapy effect of L-OHP and HT would kill the tumor cells, the mechanism was related to produce excess ROS and induce abnormal autophagy2.1 Combination therapy effect of L-OHP and HT promoted the expression of ROS.The ROS level was detected by flow cytometry. Compare with control group, the ROS level of the HT group, chemotherapy group and chemotherapy+HT group were significantly increased (P<0.05). The increase of cell viability in chemotherapy+HT group was the highest (P<0.01). the statistical result also shown that the interaction between oxaliplatin and temperature was no significant in the suppress the cell proliferation (F=3.006, P=0.118), and the main effect of oxaliplatin and temperature were significant in the suppress the cell proliferation (F=1837.156, P=0.000; F=42.392,P=0.000).The result is consistent with MTS result.2.2 Combination therapy effect of L-OHP and hyperthermia were reduced the mitochondrial membrane potentialThe MMP were analyzed by flow cytometry. Compared with control group, the MMP of the HT group, chemotherapy group and chemotherapy+HT group were declined. The MMP level of chemotherapy group and chemotherapy+HT group were significantly decreased (P<0.01).the statistical result also shown that the interaction between oxaliplatin and temperature was significant in the suppress the cell proliferation (F=70.039, P=0.000), and the main effect of oxaliplatin and temperature were significant in the suppress the cell proliferation (F=1248.867, P=0.000; F=774.044, P=0.000)2.3 Combination therapy effect of L-OHP and HT were promoted SGC-7901 cell apoptosis.The apoptosis of SGC-7901 cell was detected by flow cytometry. Compare with control group, the HT group had no significant effect on the apoptosis of SGC-7901 cell, which showed that hyperthermia treatment had only inhibition on the cell viability and had not effect on the apoptosis of SGC-7901 cell. But treated with L-OHP or L-OHP+HT had obviously effect on the apoptosis of SGC-7901 cell (P<0.01), and the lethal effect on therapy with L-OHP+HT was the most, the statistical result also shown that the interaction between oxaliplatin and temperature was significant in the suppress the cell proliferation (F=388.733,P=0.000), and the main effect of oxaliplatin and temperature were significant in the suppress the cell proliferation (F=13553.926, P=0.000; F=402.304,P=0.000)2.4 Combination therapy effect of L-OHP and HT led to autophagic cell deathIn order to study whether the apoptosis induced by the L-OHP and HT treatment was relation to the autophagic, we observed the autophagosome produced by transmission electron microscope. The result shown that the cells appeared chromatin pyknosis and autophagosome increased in the L-OHP or Chemotherapy +HT group, and the cell appeared cell membrane incompleted and were close to die in the Chemotherapy +HT group, which declared that the lethal effect on therapy with Chemotherapy +HT were relation to the autophagic.2.5 Combination therapy effect of L-OHP and hyperthermia had promoted the expression of autophagy-related protein of Beclin 1, LC3B and mTOR.The expression of autophagy-associative protein of Beclin 1, LC3B and mTOR was detected by western blot. The result shown that the expression of Beclin 1, LC3B was significantly increased in the L-OHP or L-OHP+HT group, and the expression of mTOR had negative correlation with the degree of autophagic, which demonstated that autophagy-related protein of LC3 were activated by type Ⅰ translation into type Ⅱ, the expression of Beclin 1 was significantly increased, and the expression of mTOR was significantly decreased in the L-OHP or Chemotherapy +HT group. The result further shown that the cell apoptosis was related to autophagy. the statistical result also shown that the interaction between oxaliplatin and temperature was significant in the suppress the cell proliferation, and the main effect of oxaliplatin and temperature were significant in the suppress the cell proliferation.3. Using animal tumor model to demonstrate that Combination therapy effect of L-OHP and HT could lead to autophagic cell death3.1 Established gastric cancer modelThe result showed that 5×106SGC-7901 cells were subcutaneously injected into the inguinal region of BALB/C-NU nude mouse, gastric cancer model was successfully established.3.2 Combination therapy effect of L-OHP and HT were promoted ROS producedIn the gastric cancer model, the ROS level was detected by flow cytometry. The result showed that the ROS level of the HT group, chemotherapy group and chemotherapy+HT group were significant increased in the tumor tissue compared with control group. the statistical result also shown that the interaction between oxaliplatin and temperature was no significant in the suppress the cell proliferation (F=4.573, P=0.065), and the main effect of oxaliplatin and temperature were significant in the suppress the cell proliferation (F=34.687, P=0.000; F=116.970, P=0.000)3.3 Combination therapy effect of L-OHP and HT had promoted the expression of autophagy-related protein of Beclin 1, LC3B and mTOR and tissue apoptosisThe expression of autophagy-related protein of Beclin 1, LC3B and mTOR was detected by western blot and the autophagy-related proteins of Beclin 1, LC3B and mTOR were detected by IHC. The result shown that the expression of Beclin 1, LC3B and mTOR in the L-OHP group, HT group and chemotherapy +HT group were significant increased in the tumor tissue compared with control group. The results wereconsistent with the results detected on cell level.the statistical result also shown that the interaction between oxaliplatin and temperature was significant in the suppress the cell proliferation, and the main effect of oxaliplatin and temperature were significant in the suppress the cell proliferation.The tissue apoptosis-induced DNA fragmentation was determined using the TUNEL assay. The result shown that the apoptosis level in the L-OHP group, HT group and Chemotherapy +HT group were significant increased in the tumor tissue compared with control group. The results were consistent with the results detected on cell level.Conclusion1. The ROS could downregulate the expression of m-TOR and upregulate the expression of of Beclin 1 and LC3B, there was a positive correlation between ROS level and autophagy level.2. The ROS level, induced by thermo-chemotherapy treatment, and the apoptosis of gastric carcinoma cell exist linear relationship, the result show that the ROS level play the importment role in induced the autophagic death of the gastric carcinoma cell.3. The ROS level, induced by thermo-chemotherapy treatment, could induce the autophagic death of the gastric carcinoma cell, the mechanism play the importment role in thermo-chemotherapy killed gastric carcinoma cell. |
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