| Objective:Observe and discuss the mechanism of treatment of recombinant human endostatin (Endostar) on malignant effusion action, observe and evaluate clinical efficacy and safety of Endostar in local intravesical instillation therapy for patients with malignant effusion.Methods:Choose BALB/C mice, each half of the male and female, aged4-6weeks, weighing18g-20g, reared in the SPF environment. Establish H22ascites tumor model.(1) Experimental of Endostar on mice bearing H22ascites tumor, animals groups:180healthy BALB/C mice were randomly divided into six groups, each30, Ⅰ groups: negative control group (0.9%sodium chloride,0.2ml); Ⅱ groups:the positive control group (cisplatin lmg/kg); Ⅲ groups:low-dose treatment group (Endostar5mg/kg); Ⅳ group:middle dose group (Endostar10mg/kg); Ⅴ groups:high-dose treatment group (Endostar15mg/kg);Ⅵ group:combined treatment group (cisplatin dose lmg/kg+Endostar10mg/kg). Intraperitoneal injection of medication every other day, a total of three times, before each dose were sacrificed six experimental rats. The mice were observed general, recorded weight changes, survival situation, calculate the survival rate and draw the survival curves, collect ascites and blood, use ELISA to detect VEGF in it.(2) Study of Endostar on mice with H22peritoneal ascites tumor microvessel.60mice were divided into six groups,10mice were randomly in each group. Grouped as follows:Ⅰ groups:negative control group (saline,0.2ml); Ⅱ groups:the positive control group (cisplatin, lmg/kg); Ⅲ groups:low-dose Endostar group (5mg/kg); Ⅳ group:middle dose Endostar group (lOmg/kg); Ⅴ groups: high-dose Endostar group (15mg/kg); Ⅵ group:the combination group (cisplatin 1mg/kg+Endostar10mg/kg). Mouse ascites be confirmed by ultrasound examination after grow, the above groups were intraperitoneally drug injection every other day, adjustment the drug concentration, a total of three administrations. Mice were sacrificed1day after withdrawal. Peritoneal specimens were examined by light microscopy HE staining, observe cell morphology and peritoneal vascular changes under light microscopy.(3) To detect microvascular biological factors of Endostar on mice with H22peritoneal ascites tumor.40mice were divided into two groups, the dose continuous treatment group (continuous group) and the dose administered every2days groups (every2nd group) both20; Experiments using te administered middle dose group10.00mg/kg, continuous administration group was d7, d8, d9, d10, d11, d12administration. Every2nd group was d7, d10administration.10mice were sacrificed on d7, d13. Detect expression of VEGF, MMP-2, MMP-9, HIF-1α, Survivin, Caspase-3, Caspase-9, Bcl-2and Bcl-xl in ascites and peritoneal tissue by ELISA.(4) Study of mechanism of Endostar on mice with H22peritoneal ascites tumor microvascular.78mice were divided into four groups, continuous groups (2a): d7, d8, d9, d10, d11, d12; every other group (2b):d7, d9, d11; every2group (2c):d7, d10; weekly group (2d):d7;6mice of continuous group, every other group were sacrificed on d7, d9, d11, d13, every2group on d7, d10, d13; each week group on d7, d13. Real-Time PCR to detect MMP-2, VEGF and Survivin mRNA in peritoneal tissue, western blot to detect expression levels of peritoneal protein.(5) Clinical observation on regional perfusion treatment of Endostar on malignant serous effusions. The clinical data of60cases of patients with advanced malignancies effusion, according to randomized treatment sequence, divided into Endostar perfusion chamber monotherapy treatment group (monotherapy group)31cases, Endostar local infusion in combination with cisplatin therapy group (combination group)29cases. Ultrasound-guided percutaneous drainage point positioning,"Pig tail" fluid lumen catheter indwelling catheter technique,24～48hours to do as much as possible effusion drainage, Monotherapy group gave local regional perfusion of Endostar though catheter, Pleural effusion or pericardial effusion, Endostar single injection of30mg; Peritoneal effusions, Endostar single injection of45mg, every other day, continuous use of1-2weeks, followed by an increase as the fluid, use1to2times a week to maintain. The combined treatment group patients on the basis of monotherapy combined with local topical application plus cisplatin (combination group), Cisplatin dose of30～40mg/m2, cisplatin used in the24hours after the first Endostar reperfusion. RECIST1.1standard to evaluate unmeasured lesions, calculate clinical benefit rate (DCR), evaluate the quality of life (QOL) and adverse NCI CTC3.0version of the standard to evaluate toxicity reactions.Results:(1) Experimental of Endostar on mice bearing H22ascites tumor. Before mice bearing H22ascites tumor generate ascites (1-6days), mice were eating, drinking, defecation and other activities generally good, Mice hair less shiny, responsive, slight abdominal swelling, but the extent is not obvious; Started about6-7days after inoculation, the rapid increase of mice body weight in saline control group; on the9-10day, the gradual emergence of tumor-bearing mice listlessness, sluggishness, lack of exercise, activity slow, poor response, decreased appetite, constipation, etc, ascites growing faster than each treatment group; on the11-12day late sluggish mouse move less, extreme abdominal distension, was cachexia state; on the12day mice in the control group were all died; Endostar with cisplatin group gained weight slowly, good spirit, with a median survival of17days. Inhibition of Endostar on mice with H22ascites tumor shows:on the experiment9th day, Endostar each dose group and cisplatin group (Ⅱ group) compared with the saline group(Ⅰ group) appears to reduce the amount of ascites, tumor-bearing mice were statistically significant differences in body weight(p<0.05), but Endostar each dose group than in the cisplatin group (Ⅱ group) weight difference between tumor-bearing mice was no significant difference,(p>0.05); on the11th day, weight of tumor-bearing mice was statistically significant different in Endostar each dose group than the saline group (Ⅰ group) and cisplatin group(Ⅱ group)(p>0.05); comparison between each dose group, weight of tumor-bearing mice in dose Endostar group (Ⅳ group) was statistically significant different than low dose, high dose Endostar group (Ⅲ, Ⅴ group)(p<0.05); On the9th day, the11th day, the13th day tumor-bearing mice in the combination group (Ⅵ group) compared with the saline group (Ⅰ group) were statistically significant differences in body weight(p<0.05); compared with cisplatin there are (Ⅱ group) weight difference statistically significant(p<0.05); in Each dose group, there were statistically significant differences in body weight(p<0.05). Impact on survival time of Endostar on mice bearing H22ascites tumor display:Saline group (Ⅰ group), the median survival time was10.54±0.32days, the shortest time; longest combination group (Ⅵ group), the median survival time was17.01±0.31days, Endostar different doses of tumor-bearing mice prolonged survival time compared with the control group, the difference was statistically significant(p<0.05); Endostar comparison between each dose group, survival time of dose group (IV group) was statistically significant different than low-dose group (Ⅲ group)(p<0.05); Combination group (Ⅵ group) compared with the saline group (Ⅰ group), cisplatin group (Ⅱ group), and Endostar prolonged survival in each dose group(p<0.05). Impact of Endostar on expression of VEGF in ascites and serum of mice with H22ascites tumor:on the experimental9th day, Endostar each dose group ascites and serum VEGF compared with the saline group (Ⅰ group) have statistical significance(p<0.05); between each dose group, Endostar dose group (Ⅳ group), VEGF in ascites values less than the low-dose group, high dose group (Ⅲ, V group), difference was statistically significant(P<0.05); on the11th day, each group of mice ascites VEGF values decreased significantly, were statistically significant(p<0.05); Tumor-bearing mouse serum VEGF expression in a dose Endostar group (Ⅳ group), high-dose Endostar group (Ⅴ group) compared with cisplatin group (Ⅱ group) was statistically significant(p<0.05); Endostar comparison between each dose group, values of serum VEGF was statistically significant(p<0.05); on the9th day,11th day, combined group (Ⅵ group) ascites and serum VEGF expression compared with normal saline group (Ⅰ group), cisplatin group (Ⅱ group), Endostar each dose group, were statistically significant decrease(p<0.05).(2) Study of Endostar on mice with H22peritoneal ascites tumor microvessel. Observed in all groups of mice peritoneum and abdominal viscera no obvious surface of tumor metastasis, metastatic nodules formed; Each group of mice was observed abdominal internal organs, mesentery, peritoneum membrane surface portion of the visible color than red, accompanied by a similar congestion, edema-like changes in the structure of the membrane surface appearance and no bleeding, oozing, ulceration, necrosis and other phenomena occurs in tumor-bearing mice peritoneal surface, mesenteric, and there was no bowel adhesions, bowel necrosis and so on. Light microscopic examination of each treatment group, there was no significant difference in peritoneal vessels and cell morphology, no large decrease in interstitial vascular phenomenon in cell necrosis treatment group. Peritoneal microvessel density(MVD) assay dose Endostar group13.96±0.59, compared with cisplatin group14.24±0.40and saline control group14.36±0.57, which is slightly lower MVD absolute values, but no significant difference(p>0.05). Peritoneal electron microscopy showed that the saline group peritoneal capillary permeability was significantly enhanced, fully formed from a single layer of capillary endothelial cell junctions, cell basement membrane structural damage, capillary walls thin, loose connections between cells and seen a lot of mitochondrial swelling, vacuolization phenomenon; Three different doses of Endostar group found peritoneal capillary wall thickening, and some become normal; vascular endothelial cells is small, increased nuclear heterochromatin; tight junctions between cells (gap junctions more); Inner mitochondrial edema reduced, and seen more normal mitochondria. After application Endostar prompt treatment, mouse peritoneal microvascular damage mitigation, to normalize change. Cisplatin mouse peritoneal capillary wall is thinner, heavier cell basement membrane damage; loose connection between the cells and prolonged survival in mice due to compensatory mitochondrial eventually converted to an increase in the number of mitochondria edema phenomenon; Combination therapy peritoneal capillary wall thickening; closely connected, and normal mitochondria and other vascular normalization change between cells.(3) To detect microvascular biological factors of Endostar on mice with H22peritoneal ascites tumor. Levels of VEGF, MMP-2, MMP-9, Survivin, Caspase-3, Caspase-9, HIF-1α and Bcl-2in malignant ascites of tumor-bearing mice before drug intervention were significantly higher after treatment, after Endostar intervention, indicators were significant decline, that Endostar can significantly reduced expression levels of biological factors above8in peritoneal effusions. Compare to the mode of treatment, the choice of drugs administered every other day compared with continuous administration, every expression levels of VEGF, MMP-2, MMP-9, Survivin, Caspase-3, Caspase-9, HIF-la and Bcl-2in ascites of the2nd administered group were decrease lower, compared with statistical significance between the two groups. In comparison to the staining of the peritoneal tissue, also appeared with similar results of ELISAtest in ascites, studies show that in peritoneal tissue, VEGF, MMP-9, Survivin, Caspase-3, Caspase-9and Bcl-2and other indicators appear as strong expression(+++) before treatment, MMP-2, HIF-lawas positive(++), BCL-XL rendered weakly positive(+), after Endostar intervention, MMP-2, MMP-9, HIF-la, VEGF, Survivin, Caspase-3and Bcl-2and other seven indicators come down significantly, and Caspase-9, BCL-XL showed no significant change. In comparison of the treatment, the choice of drugs administered every other day compared with continuous administration, the extent and level of MMP-9, HIF-1α, VEGF, Survivin, Caspase-3, Bcl-2and other six indicators in peritoneal tissue reduced more obvious in administered every other day, can significantly decrease the expression of negative(-) or weak expression(+). The MMP-2, Caspase-9, BCL-XL showed no difference in the two treatment modalities.(4) Study of mechanism of Endostar on mice with H22peritoneal ascites tumor microvascular. Endostar dose administration using different delivery time to detect MMP-2, VEGF, Survivin mRNA and protein expression levels in tumor-bearing mice peritoneal tissue. Endostar continuous dosing, dosing every other day, every two days and every week affecting relative expression of MMP-2mRNA, VEGF mRNA, Survivin mRNA in tumor-bearing mice peritoneal tissue shows, the relative expression was significantly decreased, with significant difference(p<0.05). Indicated that Endostar can significantly decrease expression of MMP-2, VEGF and Survivin mRNA in tumor-bearing mice peritoneal tissue, analysis and compare suppression, reduced levels in each dose group, suggesting that the group administered every2days to relative expression of MMP-2mRNA, VEGF mRNA and Survivin mRNA downward the optimal, respectively, significantly stronger than the continuous dosing group, every other day and every week dosing group, with a significant difference(p<0.05). Prompt administered every2days group is the best. This research trends similar to the overall trend in the study of MMP-2, VEGF and Survivin protein expression, but the downward effect of MMP-2, VEGF and Survivin in Endostar continuous administration group protein was significantly better than administered every other day, every2days and weekly dosing group, with statistical significance(P<0.05), prompted successive administration has a greater therapeutic benefits and treatment effect;effects of dose Endostar administered every other day group and administered every two days group were similar, no significant difference between groups(p>0.05), but the role of the two groups were better than weekly dosing group, with significant difference(p<0.05).(5) Clinical observation on regional perfusion treatment of Endostar on malignant serous effusions. The whole group of60patients completed treatment were1-5cycles, averaging2.27treatment cycles, the average duration of treatment was56.7days, all patients completed a total of136treatment cycles and can be clinically evaluated. Successful treatment of patients, tolerability satisfied, there is no adverse drug reactions in patients withdrew from the clinical treatment. Evaluation All patients recently:CR:10.00%, NCR/NPD:58.33%, PD:31.67%, DCR:68.33%, a subgroup analysis, the combined group for each single drug combination index were:6.45%,54.84%,38.71%,61.29%and19.77%,62.07%,24.14%,75.86%, and its CR, NCR/NPD and the absolute value of DCR and other indicators were significantly better than monotherapy group, analysis of variance, no significant difference between the two groups(p>0.05).QOL after treatment, all patients had varying degrees of improvement, monotherapy group QOL improved in17cases, accounting for54.84%; QOL stable in8cases, accounting for25.80%; QOL lower in6cases, accounting for19.35%. Each index corresponds to the combined group of18cases, accounting for62.07%; six cases, accounting for20.69%; five cases, accounting for17.24%. QOL comparison between the two groups was no significant difference(p>0.05), in addition, QOL improvement was also similar with the corresponding objective response in each group. All patients before and after treatment, temperature, heart rate, respiration, blood pressure and other vital signs showed no significant change. III+IV degree of toxicity appear in patients combined with cisplatin, mainly hematologic toxicities, including leukopenia, neutropenia decreased, thrombocytopenia, and chemotherapy drugs induced nausea and vomiting, fatigue and other reactions, mainly its occurrence rate was17.24%(5/29),13.79%(4/29),10.34%(3/29),20.69%(6/29) and17.24%(5/29). And no obvious monotherapy group III+IV degree of toxicity occur.Conclusion:(1) Endostar has the effect of suppressing the generation of malignant ascites and prolong survival of tumor-bearing mice, the middle dose group showed better results compared to other single dose groups, Endostar combined with cisplatin has obvious synergies effects.(2) Endostar down-regulating expression of VEGF in tumor-bearing mice ascites and blocking angiogenesis, inhibiting the formation of malignant ascites, this effect better performance in the application of the middle dose Endostar group, and may combined with cisplatin has obvious synergies effects.(3) Endostar reduced tumor-bearing mice peritoneal microvessel density absolutely, improving microvascular ultrastructure of peritoneal, reducing membrane vascular permeability, repair damage structure of the basement membrane, tight connections between cells, improving mitochondrial swelling, vacuolization state, triggering a compensatory increase in mitochondria, promote the normalization of blood vessels. This effect administered in combination with cisplatin and Endostar more obvious manifestations, suggesting a synergistic effect between them.(4) Endostar may reduce expression of VEGF, MMP-2, MMP-9, Survivin, Caspase-3, Caspase-9, HIF-la, Bcl-2and other tumor-related factors in ascites and peritoneal tissue, reducing vascular permeability, maintaining the integrity of the basement membrane, inducing tumor cell apoptosis and improve tumor hypoxia microenvironment, inhibit the generation of malignant effusion. Further, there was a certain difference between this effect and the different methods of administration.(5) Endostar can significantly reduce the expression levels of MMP-2, VEGF, Survivin mRNA and protein expression levels in tumor-bearing mouse peritoneal tissue. Mechanism of generation of malignant effusion and application of Endostar is the result of combined effects of multiple factors. Endostar continuous, every other day, every2days dosing was significantly better than the effect of the mode of every week administration.(6) Endostar regional perfusion can better improve in patients with malignant effusion recent treatment, patient tolerated satisfactory and has a good clinical safety, Endostar intravesical instillation of chemotherapy drugs may have the potential synergy effects. |
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