Curative Effect And Safety Analysis Of 5-fluorouracil Aerosol Chemotherapy For Intra-abdominal Implantation Of Gastric Carcinoma In Nude Rats

BackgroundGastrointestinal cancer is one of the most common malignant tumor in our country, and most are in advanced stage when diagnosed. After underwent surgical resection combined with chemotherapy and radiotherapy, about a half of patients experienced tumor recurrence and/or metastasis within 5 years, which directly affects the long-term survival of patients. The presence of free cancer cells (FCC) and micro-foci is one of the main causes of tumor recurrence and metastasis. In recent years, laparoscopic surgery was gradually applied to gastrointestinal cancer resection dute to the advantages of minimal invasivenses, reduced postoperative pain and improved recovery. Usually pneumoperitoneum is essential establish for vision exposure during laparoscopic surgery, but the FCC may spread to the entire abdominal cavity and adhere to the surface of ograns via eddy effect of the media of pneumoperitoneum, resulting in widespread implantation and/or metastasic. Domestic and foreign scholars had done a lot of research to prevent tumor recurrence and metastasis of gastrointestinal cancer after surgery, such as preoperative intra-arterial infusion chemotherapy, enteric cavity chemotherapy during operation, preoperative radio-chemotherapy, adjuvant chemotherapy and et cetera. Recent years, intraperitoneal chemotherapy (IPC) with the promising effect to prevent and treat the tumor implantation and/or metastasis after surgery has became a research hotspot because of its distinct pharmacokinetic characteristics and other basic characteristics. Study showed that anti-tumor effect of chemotherapeutic drugs depend on the concentrations of drugs around tumor lesion. After intraperitoneal administration, drugs entered the liver via portal vein and degradated in the liver and only a few entered body circulation after peritoneal clearance. Unlike intravenous chemotherapy through systemic clearance, drug concentrations in the abdominal cavity can be several or even dozens of times higher, due to the "abdominal cavity plasma barrier", providing a constant sustained high concentrations of chemotherapy drugs in which the FCC and micro-foci were directly soaked, and the anti-tumor effect were greatly enhanced. Therefore, we envisaged a new therapeutic approach, that's proceding the aerosol chemotherapy and laparoscopic surgery simultaneously, combining the characteristics of laparoscopic pneumoperitoneum with IPC to kill the FCC safely and effectively. To evaluate the feasibility, curative effect and safty of 5-fluorouracil aerosol intraperitoneal chemotherapy on gastric carcinoma, we used self-developed aerosol chemotherapy instrument to conduct research in vivo and vitro.Objectives1. To developed an aerosol chemotherapy instrument, which can transform the liquid medication into aerosol and maintain its chemical nature, and produce certain pressure to facilitate perfusion.2. To evaluate the effect of mimic 5-fluorouracil (5-Fu) aerosol intraperitoneal chemotherapy (AIPC) on the human gastric cancer cell line MKN-45 in vitro.3. To evaluate the curative effect and safety of 5-fluorouracil (5-Fu) aerosol intraperitoneal chemotherapy (AIPC) for intra-abdominal implantation of gastric carcinoma in nude rats.Methods1.Development of aerosol chemotherapy instrument.Use a specific ultrasonic frequency in accordance with the molecular weight of the Chemotherapy drugs (5-fluorouracil) to transform the liquid medication into aerosol and maintain its chemical nature, and control the aerosol volume produced per unit of time in the meantime.2. Mimic aerosol intraperitoneal chemotherapy in vitro.Create an aerosol pressure bottle:Use a 1 L suction bottle with two small holes on the cover, one connecting with aerosol chemotherapy device and the other connecting with pneumoperitoneum machine for pressure monitor, and then make one more hole as a vent hole.3. MKN-45 human gastric cancer cells.MKN-45 was cultured in the cultivation of RPMI-1640 (containing 10% fetal calf serum and a final concentration of 100 u/ml penicillin and streptomycin) at 37℃,5% CO2.4. Experimental animal.A total of 20 male nude rats at age of 10 weeks with a weight of 200-230 g, supported by the Shanghai Public Health Clinical Center were kept in Southern Medical University Experimental Animal Center. All nude rats and experimental conditions achieve specific pathogen-free (SPF) standard.5. Detection of inhibition of cell proliferation by MTT assaySingle-cell suspension of logarithmic growth phase of MKN-45 cell were plated in 96-well culture plates with 4×103/ml per well in a 5% CO2 incubator at 37℃. When entered the logarithmic growth phase, cells were divided into 3 groups: negative control group,5-Fu aerosol group and normal saline group.5-Fu aerosol group:cells were placed in the aerosol pressure bottle (pre-placed in UV cabinets for 60 min for disinfection), connected with aerosol chemotherapy instrument, and treated with 0.1 mmol/L 5-Fu aerosol for 30 min when adjust the aerosol pressure at about 8 mmHg; NS group were treated with normal saline aerosol and the pressure and time were the same as 5-Fu aerosol groups. After medium was added, each group was placed in 37℃,5% CO2 incubator and cultured for 24h before disposable culture medium, and added 5mg/ml of MTT to each well, after culture for another 4 h, culture medium and MTT were sucked out, and each group was added with DMSO 150μl, then placed on microplate oscillator for 10 min before OD value were measured by enzyme-linked immunosorbent assay at absorbance of 490 nm and calculated the inhibition rate of cell proliferation. The rate (%)= (1-OD value of treated group/OD value of the control group)×100%. The entire experiment was repeated 3 times.6. Detection of apoptosis by FCM.Single-cell suspension of logarithmic growth phase of MKN-45 cell were plated in 96-well culture plates with 4×103/ml per well in a 5% CO2 incubator at 37℃. After serum-free RPMI-1640 medium synchronization, cells were divided into 2 groups:5 -Fu aerosol group and NS aerosol group. After the treatment medium were added into each well for 24 h, cells were collected, washed with PBS, added with 500μl of BindingBuffer for cells suspension, and then added with 5μl AnnexinⅤ-FITC, mixed, then added with 5μl Propidiumlodide, mixing at room temperature, dark reaction for 15 min, using flow cytometry to detecte the proportion of apoptotic cells.7. Detection of cell cycle by FCM.Single-cell suspension of logarithmic growth phase of MKN-45 cell were plated in 96-well culture plates with 4×103/ml per well in a 5% CO2 incubator at 37℃. Serum-free RPMI-1640 medium synchronization, groups and treatment were the same with above, after the treatment medium were added into each well to cultured for 24 h, then cells were collected, washed with PBS and counted, adjusted to 1×106/ml single-cell suspension, fixed with 70% ethanol, preserved at 4℃, then wash with PBS before staining. After added 100μl RNase A and treated with 37℃water bath for 30 min,400μl PI staining mixture was added and incubated at 4℃dark for 30 min, then determine the percentage of each cell cycle according to the relative DNA content of cells with different distribution.8. Establishment of the animal model of nude rats with petitoneal implant of human gastric carcinoma (MKN-45) cell.Nude rats were fasted for 12 h and intraperitoneally injected with preparation of gastric cancer cell MKN-45 cell suspension 2ml (5 x 107/ml), before randomly divided into two groups (10 in each), then treated with aerosol intraperitoneal chemotherapy 2 hours later.9. Aerosol intraperitoneal chemotherapy in nude rats.Nude rats were anesthetized by inhalation and fixed on a surgical operation table. Two veress needles were inserted into left lower quadrant in nude rats, one connecting aerosol chemotherapy device while another connecting pneumoperitoneum machine for monitoring the insufflation pressure. An infusion tube was inserted into the right lower abdomen to adjust the gas flow to maintain the gas abdominal pressure at about 8 mmHg. Experimental group:the 5-Fu (600mg/m2) diluted with normal saline 20 ml was perfused into the abdominal cavity. Control group:perfused with NS aerosol of normal saline 20ml. Both two group were treated for 30 minutes.10. Observation and measurement.φGeneral situation including mental state of rats, activity, appetite and the changes of weightwas recorded weekly; 2 Physiological and biochemical parameters:after 2 ml venous blood of all nude rats were phlebotomized for blood biochemical tests on one day before the intraperitoneal injection of gastric cancer cells and one week after aerosol intraperitoneal chemotherapy, parameters including red blood cell (RBC), hemoglobin (HB), white blood cell (WBC), platelets (PLT), alanine aminotransferase (ALT), aspartate aminotransferase (AST) were tested; (?) anatomy:all nude rats were executed 5 weeks after the experiment, dissected and exposed to observe whether there were ascites, foci that can be transfered to the liver, spleen, peritoneum and other organizations were collected and dehydrated by 4% formaldehyde before HE staining; 4 tumorigenic rate.11. Statistical analysisAll the statistical analyses were carried out with SPSS 13.0 software and measurement data were presend as x±s. The difference between mean of two independent samples were analyzed by Student t-test and changes of physiological and biochemical parameters were analyzed by analysis of covariance. Difference between numeration data were analyzed by x2 test. Body weight changes were analyzed by repeated measures analysis of variance and P<0.05 was considered significant.Results.1. Inhibition of the cell proliferation of gastric cancer cells by 5-Fu aeorsol:the inhibiton rate of the cell proliferation of 5-Fu aerosol group was 31.13%, as the NS group 4.65%, the difference between the two groups was statistically significant, (P <0.001);2. Ratio of apoptosis induced by 5-Fu aerosol:the ratio of apoptosis induced by 5-Fu aerosol group was 12.00%, significantly higher than the NS group with 2.65%, the difference between the two groups was statistically significant, (P<0.001); 3. Cell cycle of MKN-45 gastric cancer cell affected by 5-Fu aerosol:compared with the NS group, the G1 and G2 stage proportion of aerosol 5-Fu group were significantly increased[(51.83±1.95)%vs(36.41±2.33)%,P<0.001; (31.45±1.69) %vs (18.39±2.10)%, P<0.001], and the S stage proportion of aerosol 5-Fu group was significantly reduced[(16.72±2.36)%vs (45.20±3.27)%, P<0.001].4. General situation of the nude rats:no nude rats died during the experiment, the initial period after aerosol intraperitoneal chemotherapy, nude rats of the two groups showed listlessness, lethargy, unresponsive and activities reduction, no vomiting and diarrhea. After a few hours the symptoms gradually reduced and returned to normal;5. Tumorigenic rate:the rate of the experimental group and control group was 40% and 100%, compared the two groups, x2= 8.57, P= 0.003, the difference between the two groups was statistically significant;6. Changes of weight:there were significant differences between the experimental group and control group (P<0.001), before aerosol chemotherapy, the weight of the experimental group nude rats was 219.67g, and the control group was 220.33g, which showed no significant difference (P= 0.888),2 weeks after aerosol chemotherapy, the weight of the experimental group was 258.65g, and the control group was 254.78g, there was no significant difference between the two groups (P= 0.780),3 weeks after aerosol chemotherapy, the weight of the experimental group was significantly greater than the control group, which was 275.67g vs 261.62g (P= 0.007),5 weeks after aerosol chemotherapy, the weight of the experimental group was 301.20g, which was still significantly higher than 279.55g in control group(P= 0.035);7. Changes of blood physiology and blood biochemistry results:the values such as WBC, RBC, HB, PLT, ALT and AST of the two groups before and after aerosol intraperitoneal chemotherapy showed no significant difference (P> 0.05);8. Anatomy:there were no obvious ascites in both two group of nude rats, tumor nodules mainly distributed in the intestinal serosa and the abdominal wall puncture site of the tumor cell injection, looks gray, size range, some with peritoneal mesenteric. It is poorly differentiated adenocarcinoma and constitute of a great quantityinfiltrative growth tissue observation by light microscope, much to rank nest-form, and some cryptae sample structure. Cancer cell looked inequality of size, heteromorphism obviously,much have caryocinesia image.Conclusion1.5-Fu aerosol can inhibit the proliferation, induce apotosis and block the cell cycle of G1 stage of gastric cancer cell effectively;2.5-Fu AIPC can prevent and treat the intra-abdominal implantation of gastric carcinoma effectively and safety which providing a new useful method for killing the free cancer cells during laparoscopic surgery, and is expected to provide the experimental basis for the clinical application of AIPC with laparoscopic surgery simultaneously.