Abstract Objective To compare and pirarubicin (Pirarubicin, THP) and other chemotherapy drugs, the synergistic effect of gastric carcinoma warm synergy mechanism of chemotherapy drug Hyperthermic Chemotherapy. Methods 36 cases of resected tissue, the activity of human gastric tissue in vitro three-dimensional culture role model, MTS PMS colorimetric detection of tissue culture in order to understand the antitumor effect of chemotherapy drugs Hyperthermic Chemotherapy. TUNEL fluorescence detection in situ apoptosis, the HE staining study evaluation of drugs on the gastric tissue changes in tissue morphology and electron microscopy. Gastric carcinoma, DDP, MMC and THP destruction have strong warm collaborative relationship (P = 0.000), the THP separate processing and warm co-processing has a strong inhibitory effect, and the other three drugs inhibit the rate with a significant difference (P <0.05). 0.05）。">THP and warm effect of co-processing and gastric cancer cases histological grading (P = 0.000), and has nothing to do with age, gender, tumor size, clinical stage and preoperative CEA levels (P> 0.05). The THP warm co-processing apoptotic index and the other three drugs have significant differences (P <0.05). HE staining showed that THP and warm treatment, gastric carcinoma most of disintegration, cell degeneration and necrosis. Electron microscopic analysis showed that co-processing by the THP and warm invagination of the cell membrane, cytoplasmic vacuoles, and chromatin concentration, while the nuclear membrane disappears. Conclusion THP gastric carcinoma has a good warm synergies and anti-tumor effect, further clinical research value.
Key words pirarubicin star warm effect gastric
Gastrointestinal malignancy is the most common malignant tumors of the clinical, surgery is still the preferred method for the treatment of gastrointestinal tumors, but still have a higher recurrence rate after surgery for advanced gastrointestinal cancer, radical. Intraoperative intraperitoneal hyperthermic perfusion chemotherapy (Itraoperative peritoneal hyperthermia chemotherapy, IPHC) direct role in the recurrence and metastatic sites, a role in the prevention of gastrointestinal cancer after radical resection of recurrence, and side effects are small, at home and abroad in recent years research one of the hotspots. However, infiltration subperitoneal deeper tumor the HIPEC chemotherapy still peritoneal recurrence. Therefore, the research and application of penetrating good drugs to improve drug penetration ability, enhance the osmotic effect of the heat will be the direction of future research. This study intends to establish a gastric carcinoma small three-dimensional culture and technology, research pirarubicin Star (Pirarubicin, THP) in gastric carcinoma warm synergies and mechanism of action.
1 Materials and methods
1.1 gastric cancer specimens from 2004 to 2005, hospital underwent radical gastrectomy resection or gastric surgical specimens of 36 cases of stomach full cut, definitive diagnosis were confirmed by pathology. Among them, 22 males, 14 females, aged 33 to 85 years, an average of 60.7 years. Patients with clinical stage, histopathological classification, preoperative CEA levels and tumor diameter data are shown in Table 1. Table 1 gastric cancer cases background and THP warm inhibition rate
THP inhibition rate of the variable number of cases
502675.6±8.9 <501076.6±11.6病理组织学分类高分化腺癌490.6±5.5 中分化腺癌1084.8±4.7 低分化腺癌2069.9″>(± s)% Gender Male Female 22 78.1 ± 11.6 1473.5 ± 9.2 Age (years)> 502,675.6 ± 8.9 <501,076.6 ± 11.6 pathological histological classification differentiated adenocarcinoma 490.6 ± 5.5 differentiated adenocarcinoma 1084.8 ± 4.7 poorly differentiated adenocarcinoma 2069.9 2cm2875.1±11.3 ≤2cm880.5±7.8术前CEA水平（ng/ml） >512 76.5±10.7 <524 76.0±11.4临床分期（TNM">the ± 8.4 signet ring cell carcinoma of 269.0 ± 3.3 tumor diameter (cm)> 2cm2875.1 ± 11.3 ≤ 2cm880.5 ± 7.8 preoperative CEA level (ng / ml) 512 76.5 ± 10.7 <524 76.0 ± 11.4 clinical stage (TNM ) Ⅰ 585.5 ± 5.6 Ⅱ 1575.2 ± 9.5 Ⅲ 13 77.4 ± 11.0 Ⅳ 362.1 ± 9.4
1.2 chemotherapy drug cisplatin (DDP) was purchased from Qilu Pharmaceutical Factory, pirarubicin Star (THP) the Shenzhen Wanle Pharmaceutical Co., Ltd., mitomycin C (MMC) is Japan’s Kyowa Hakko, fluorouracil (5 Fu Injection ) products for Zhejiang Haipu pharmaceutical company. Drug storage solution is formulated according to the instructions, and stored at -20 ℃ refrigerator.
1.3 reagents and consumables without phenol red RPMI 1640 and fetal bovine serum products; MTS purchased from Promega; Hyclone Company the Phenazine sulfate ester (Phenazine methosulfate, PMS), DMSO, neomycin and amphotericin B were purchased from Sigma products; collagen sponge Beijing benefits and health biotechnology products and the specifications for the 10 × 1.5 × 0.1 before use sponges cut into 0.5cm size; 24-well plates for Costar products.
1.4 warm effect according to Hoffman  and other methods of fat and fibrous tissue peeling off the surface of the surgical specimens were washed with PBS the bloodstained and dirt of the surface of the specimen is then placed in the soaking solution containing antibiotics (neomycin 50μg / ml, amphotericin 5μg/ml) soaked in 15min. Further added 1640 medium for washing, the specimen is cut to 1mm size pieces as far as possible with a scalpel, the three tissue block is added to the collagen sponge, and then transferred to the pre medium 0.6ml 24 orifice holes, incubator 37 ℃ overnight. Next day, each well along the hole wall was slowly added to the drug-containing medium 0.4 ml, three parallel holes of the concentrations of each drug, in the the 42 ℃ role 1h, and then sucked out drug, washed twice in culture medium, adding fresh culture liquid 1ml culture was continued 3d. Applications reported THP Hyperthermic Chemotherapy little research references intravenous dose (40mg/m2) concentration set to 32μg/ml. The concentration of other drugs references reported , which, DDP concentration 50μg/ml MMC concentration 15μg/ml fluorouracil 350μg/ml.
The colorimetric MTS 1.5MTS PMS formulated formulated in DPBS 2mg/ml, 0.22μm membrane filter sterilization, packaging. PMS PREPARATIONS: PBS formulated into liquid storage of 100mmol / L, 0.22μm membrane filter sterilization, at 4 ° C in the dark to save. MTS PMS solution: join PMS mixed with freshly prepared MTS (2mg/ml) 20:1 volume. MTS PMS Determination method: 24-well plates each well was added 100μl of MTS PMS use solution, the culture was continued for 4h, the oscillation 10min, 100 μl per hole, the absorbance value (OD490) was measured with a wavelength of 490nm of the three parallel holes of average.
Inhibition rate (%) = (dosing hole OD-blank wells OD) / (control wells OD-blank wells OD) × 100
1.6TUNEL fluorescence detection tissue into small pieces after the treatment of the in situ apoptosis drugs placed in the the constant cold box frozen slicing machine slices shelves, frozen hardens slice, thickness of the block to be tissue as 5 to 7 μm. 4% polyoxymethylene 4 ° C fixed 30min, by 0.1% Triton X-100 on ice penetration 5min, washed 3 times with PBS and a total of 15min. FITC-labeled TUNEl reaction mixture, 37 ° C wet box reaction 90min, then washed three times with PBS, a total of 30min, 80% phosphoric acid glycerol were mounted. Take non-necrotic area fluorescence microscopy to count more than 200 cells, as positive rate statistics (apoptotic index, AI).
1.7 tissue and cell morphology study drug treatment organizations small pieces placed in 10% neutral buffered formalin the 4 ℃ fixing 48h, after a series of ethanol dehydration, paraffin-embedded sections. Sections were hematoxylin and eosin staining was observed under a microscope of hyperthermic chemotherapy before and after tumor histological changes; specimens for electron microscopy analysis of the 2% glutaraldehyde followed by oxide the Wo processing and gradient dehydration, embedding and Slice microscopy.
1.8 Statistical analyzes were performed using SPSS 10.0 statistical analysis software. Chemotherapy drugs warm synergy with chemotherapy drugs alone two groups compared using a paired t-test; role in the differences between the various chemotherapy drugs were analyzed using one-way analysis of variance, THP bivariate correlation analysis of the inhibition of gastric tissue and clinical factors correlation analysis, calculated the Spearman correlation coefficient.
2.1 hyperthermic chemotherapy drugs role of gastric carcinoma gastric cancer tissues were killing role, as shown in Table 2. Which, DDP, MMC and THP three drugs have warm synergies, the inhibitory effect of the heat treatment compared with separate processing has been enhanced (t value respectively 8.099,8.675,8.553; P = 0.000); 5-Fu no significant warm synergies (t = 0.880, P = 0.385). The THP alone processing and warm co-processing has a strong inhibitory effect, and the other three drugs inhibit the rate has a significant difference (P <0.05). Table 2 drugs on the gastric tissue of warm synergistic inhibition rate of the drug name alone
The relationship between the role of gastric cancer tissues and clinical factors 2.2THP THP THP inhibitory effect on the tumor and the age, gender, preoperative CEA levels, clinical studies have shown that inhibition of gastric cancer tissues and clinical factors (Table 1), sub- 0.05），而与肿瘤的病理学分级有关。">period and tumor size (P> 0.05), with tumor pathological grade. From well-differentiated adenocarcinoma, in differentiated adenocarcinoma with histological grade, poorly differentiated adenocarcinoma signet ring cell carcinoma, THP inhibition rates show a downward trend (P = 0.000). The 37 ℃ Spearman correlation coefficient γ = -0.479, P = 0.003; 42 ° C Spearman correlation coefficient γ = -0.843, P = 0.000. Several other chemotherapy drugs DDP, MMC, 5-Fu Whether it is in the case of 37 ℃ or 42 ℃, the inhibitory effect of the tumor and the age, gender, preoperative CEA level, clinical stage, tumor size and tumor tissue sub- 0.05）。">level were unrelated (P> 0.05).
2.3 in situ detection of apoptosis drug for gastric carcinoma has a certain role in the induction of apoptosis, in Table 3, Figure 1a, b. Which, DDP, MMC and THP three drugs have warm synergies, isothermal heat treatment than those dealt with separately has significantly increased apoptosis index (P <0.05). THP warm collaborative processing has strong apoptotic effect, apoptosis index and the other three drugs have significant differences (P <0.05).
2.4HE staining and electron microscopy analysis of HE staining showed significant killing effect, THP and warm synergy of gastric carcinoma has four drugs in Table 3 hyperthermic chemotherapy drugs induced apoptosis of gastric carcinoma name alone apoptotic index
The complete morphology tumor tissue under the light microscope vision loss, severe necrosis of the tumor cells, the nucleus pyknosis cytoplasm lost and accompanied by a large number of nuclear fragmentation phenomenon, as shown in Figure 1c, d. Electron microscopy analysis showed that THP and warm co-processing cells showed an early, middle and late apoptotic characteristics, membrane invagination, cytoplasmic vacuoles, and the emergence of the chromatin concentration, while the nuclear membrane disappears, as shown in Figure 1e, f.
1/5 of the gastric cancer accounted for half of the deaths of gastrointestinal tumors and various malignancies deaths, ranking the forefront of cancer deaths. In recent years, with the progress of surgery, postoperative lymph node recurrence has been significantly reduced, replaced by peritoneal metastasis and recurrence. How to prevent the recurrence of postoperative abdominal gastrointestinal tumor pressing problem.
Currently, intraoperative intraperitoneal hyperthermic perfusion chemotherapy has gradually become accepted by most scholars effective method of treatment of advanced tumors of the gastrointestinal tract. Hyperthermic intraoperative intraperitoneal chemotherapy in gastrointestinal tumors postoperative peritoneal metastases and recurrence achieved significant results, and can significantly improve survival, but there are many details worth exploring, such as chemotherapy drugs choice, the choice of the specific method , liquid capacity, the determination of dose, etc., are the problems to be solved.
Pirarubicin chemical structure of a tetrahydropyran group, thereby partially bypassed gp170 glycoprotein-mediated doxorubicin resistance . The study showed that the leukemia cells HL 60 THP intake very rapidly, in 1μg/ml the concentrations 30s within the THP cumulative intake 0.262μg, and the main absorption into the nucleus; doxorubicin only 0.0015μg, large portion is in the cytosol, while THP discharge slower, intracellular able to retain high concentrations of . Sugano et al  in bladder cancer cavity perfusion study shows that in the animal model, THP cancer have a strong penetration can penetrate within 60min 2 ~ 3mm tissue, but doxorubicin only penetrate 1mm . Therefore, THP hyperthermic perfusion chemotherapy have better prospects for clinical application.
At present, the international community rarely been reported about THP hyperthermic perfusion chemotherapy of basic research and clinical applications. The results of this study show the THP 42 ℃ synergistic killing effect of gastric carcinoma than commonly used drugs such as DDP, MMC and 5-Fu. The preliminary work of this study show that the co-processing of human gastric cancer MGC 803 cells THP and warm in the S phase of the cell increased the G2 / M phase cells decreased . Have
a, b are in situ fluorescent TUNEL apoptosis staining (× 200), a control without drug, b is 42 ° C + THP white arrow indicates apoptotic cells; c, and d are the HE staining (× 200), C without drug control, and d is 42 ° C + of THP; e, f are the observation results, e is of gastric cancer in the untreated cells (× 5800), and f is 42 ° C + of THP processing after the apoptosis of cells (x 7500)
Figure 1 THP and warm synergy of gastric carcinoma
The reports suggest that the destruction of cells in the G2 / M phase, THP strongest G0 / 1 cell killing effect is weak. As the warm is asynergy increase cellular uptake of THP. Thus, by isothermal treatment block the cell cycle at the G2 / M phase, is to increase the sensitivity of cells to THP important mechanism.
Clinical factors correlation analysis showed that at 37 ℃ or 42 ℃ killing effect and the patient’s age, gender, clinical stage, tumor size, and preoperative CEA levels, THP no obvious relationship with gastric cancer histopathological grading, inhibition in most well-differentiated adenocarcinoma. Foreign clinical trial studies have shown that intraoperative hyperthermic perfusion chemotherapy clinical efficacy and clinical stage gastric II and III patients, intraoperative warm infusion chemotherapy can significantly delay recurrence and prolong survival time Ⅲ more than patients, intraoperative intraperitoneal hyperthermic chemotherapy has no significant survival advantage . This study is considered local gastric carcinoma in vitro studies, the influencing factors is relatively small, and the results of clinical studies, and many clinical factors, possible side effects and complications, such as intraoperative intraperitoneal hyperthermic chemotherapy surgery are clinical efficacy influential. THP inhibition of gastric carcinoma correlated with clinical efficacy, good or bad inconsistencies are not contradictory.
Is generally believed that the low systemic chemotherapy, tumor differentiation, the effect of THP. The results of this study suggest that a high degree of tumor differentiation, THP inhibits tumor effect. This may be related to local chemotherapy drug concentration is much higher than with systemic chemotherapy drug concentrations used in this study. As the good THP permeability, penetration into the intracellular drug resistance protein concentration is much higher than the concentration of pump drugs out of tumor cells, then played the role of the resistance protein on the resist chemotherapy is relatively small, so the THP infusion chemotherapy on better resistance protein function better in well-differentiated tumor cells. Other chemotherapy drugs such as DDP, MMC, 5-Fu and histological grade non-related reasons also may be related to their permeability relatively poorer.
In summary, THP gastric carcinoma has a good warm synergies and anti-tumor effect, with intraperitoneal hyperthermic perfusion chemotherapy research value. Intraoperative Intraperitoneal Hyperthermic Chemotherapy is meaningful in the perioperative clinical select appropriate clinical stage and histological grade gastric cancer cases.