Application of ultrasonography in predicting the biological risk of gastrointestinal stromal tumors.

OBJECTIVES: To explore and establish a reliable and noninvasive ultrasound model for predicting the biological risk of gastrointestinal stromal tumors (GISTs). MATERIALS AND METHODS: We retrospectively reviewed 266 patients with pathologically-confirmed GISTs and 191 patients were included. Data on patient sex, age, tumor location, biological risk classification, internal echo, echo homogeneity, boundary, shape, blood flow signals, presence of necrotic cystic degeneration, long diameter, and short/long (S/L) diameter ratio were collected. All patients were divided into low-, moderate-, and high-risk groups according to the modified NIH classification criteria. All indicators were analyzed by univariate analysis. The indicators with inter-group differences were used to establish regression and decision tree models to predict the biological risk of GISTs. RESULTS: There were statistically significant differences in long diameter, S/L ratio, internal echo level, echo homogeneity, boundary, shape, necrotic cystic degeneration, and blood flow signals among the low-, moderate-, and high-risk groups (all p < .05). The logistic regression model based on the echo homogeneity, shape, necrotic cystic degeneration and blood flow signals had an accuracy rate of 76.96% for predicting the biological risk, which was higher than the 72.77% of the decision tree model (based on the long diameter, the location of tumor origin, echo homogeneity, shape, and internal echo) (p = .008). In the low-risk and high-risk groups, the predicting accuracy rates of the regression model reached 87.34 and 81.82%, respectively. CONCLUSIONS: Transabdominal ultrasound is highly valuable in predicting the biological risk of GISTs. The logistic regression model has greater predictive value than the decision tree model.

[Microbubbles targeted to P-selectin for evaluating testicular ischemia-reperfusion injury in rabbits].

OBJECTIVE: To explore the feasibility of evaluating complete ischemia-reperfusion injury (IRI) of the testis by contrast-enhanced ultrasonography with microbubbles (MB) targeted to P-selectin (MBp) in rabbits. METHODS: We randomly divided 30 healthy adult rabbits into five groups of equal number (control, 0.5 h IRI, 1 h IRI, 2 h IRI, and 4 h IRI), prepared phospholipid MB and MBp, and performed contrast-enhanced ultrasonography of the bilateral testes with MB or MBp at an interval of 20 min at different times after IRI. When MB or MBp disappeared completely in the healthy testis at 4 to 5 min after intravenous injection, we recorded the power of the first frame (F-P) in the IRI testes followed by immunohistochemical staining of the testis tissue. RESULTS: CEU with MBp achieved a significantly higher F-P than that with MB in all the IRI groups (P < 0.05), which was (8.34 +/- 1.20) versus (1.87 +/- 0.25) 10(-5) AU at 2 hours, but there was no significant difference between MB and MBp in the control rabbits (0 AU, P > 0.05). Immunohistochemistry showed a significantly time-dependent increase in the expression of P-selectin in the vascular endothelial cells of the IRI testes, but not in those of the control. CONCLUSION: Contrast-enhanced ultrasonography with MBp can be used to evaluate the inflammatory reaction of testicular ischemia-reperfusion injury.

Intermittent maternal hypoxia has an influence on regional expression of endothelial nitric oxide synthase in fetal arteries of rabbits.

BACKGROUND: Maternal hypoxia induces sustained fetal adaptations associated with changes in gene expression. We hypothesized that intermittent maternal hypoxia has an influence on regional expression of endothelial nitric oxide synthase (eNOS) in fetal arteries of New Zealand White rabbits. METHODS: Timed-pregnant New Zealand White rabbits (term = 30 ± 1 d) were randomly assigned to a normoxic control group (n = 5) or a hypoxia group (12% O2, n = 5) during days 10-29 of pregnancy. At the end of pregnancy (29 d gestation), blood samples were collected from mothers and fetuses. Carotid and femoral arteries of fetuses were extracted for eNOS mRNA and protein concentration and analysis of total NOS activities. RESULTS: Our data demonstrate that chronic intermittent maternal hypoxia significantly increased eNOS mRNA and protein concentrations and total NOS activities in carotid artery segments but decreased eNOS mRNA and protein concentrations and total NOS activities in femoral artery segments in the same fetuses. Vascular endothelial cells, but not smooth muscle cells, of fetal rabbits exhibited positive immunostaining for the eNOS protein. CONCLUSION: These observations suggest that chronic hypoxia can regulate regional expression of eNOS as an adaptive response to hypoxic stress in fetal arteries.

Effect of interstitial chemotherapy with ricin temperature-responsive gel for anti-breast cancer and immune regulation in rats.

OBJECTIVE: To explore the effect of ricin temperature response gel on breast cancer and its regulatory effect on immune function in rats. METHODS: Ricin was purified by chromatography and identified by immunoblotting. The rat subcutaneously transplanted breast cancer model was established. Forty model rats with a tumor diameter of about 3.0 cm were subjected to the study. They were randomized into four groups equally: the model group and three treated groups (blank gel, ricin, ricin-gel) were administered with blank gel, ricin, and ricin temperature response gel via percutaneous intratumor injection, respectively. The tumor was isolated 10 days later for the estimation of tumor inhibition rate (TIR) by weighing, pathologic examination, and detection of tumor apoptosis-associated genes bcl-2 and bax with semiquantitative RT-PCR. Also, peripheral blood was obtained to test T-lymphocyte subsets, the killing function of lymphocytes, and the contents of tumor necrosis factor-α (TNF-α) and interleukin-2 (IL-2). The outcomes were compared between groups. RESULTS: The TIR in the ricin-gel group was 61.8%, with the pathologic examination showing extensive tumor tissue necrosis. Compared with the model group, after ricin temperature response gel treatment, bcl-2 expression was down-regulated, bax expression was up-regulated, CD4+ lymphocytes and CD4+/CD8+ ratio in peripheral blood were increased, the killing function of lymphocytes was enhanced, and the contents of TNF-α and IL-2 were elevated (P < 0.05 or P < 0.01). CONCLUSION: Intratumor injection of ricin temperature-responsive gel showed significant antitumor effect on breast cancer and could enhance the immune function in the tumor-bearing rat.

Development, characterization and anti-tumor effect of a sequential sustained-release preparation containing ricin and cobra venom cytotoxin.

Cobra venom cytotoxin (CVC) loaded in poly (lactide-co-glycolide) (PLGA) microspheres was mixed with ricin and encapsulated in a thermosensitive PLGA-PEG-PLGA hydrogel for this study. This sequential sustained-release preparation (SSRP) containing ricin and CVC could avoid burst release effect of CVC from microspheres. In addition, in SSRP, the two biotoxins have different drug release rates and antitumor mechanisms, which can be complementary to each other. Ricin has a faster release rate than CVC. It can combine with the tumor cell membrane and enter the cell, inhibiting protein synthesis within 2 weeks. Whereas CVC releases slowly in 5 weeks directly dissolving the tumor cell membrane and killing the cells which are less-sensitive to ricin. The in vivo experiments showed that intratumoral injection of SSRP could inhibit hepatocellular carcinoma growth significantly, and the tumor growth inhibition rate reached 73.5%. It appears that a new medicine preparation for cancer local treatment should be further studied for clinical applications.

Chemotherapy with PLGA microspheres containing docetaxel decreases angiogenesis in human hepatoma xenograft.

To investigate the antiangiogenic effect of sustained-release poly (lactic-co-glycolic acid) microspheres containing docetaxel (PMCD) in human hepatoma xenograft. PMCD were prepared by solvent evaporation method with an encapsulation efficiency of 98.7% and a release period of about 3 weeks in vitro. PMCD were intratumorally injected once for mice bearing a human hepatocellular carcinoma. On day 21 post-treatment, the inhibition rate of tumor growth was 72.7% in the high-dose group, indicating a significant antitumor activity. Meanwhile, excellent antiangiogenic effect was observed based on the contrast-enhanced ultrasonography as well as microvessel density determination. Additionally, the real-time fluorescence quantitative PCR revealed that the expressions of vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), and angiopoietin-2 (Ang2) genes were down-regulated significantly. Interstitial chemotherapy using PMCD was highly effective and safe for the treatment of the human hepatoma xenograft and that decreasing angiogenesis could be one of the most important mechanisms involved in the antitumor activity.

Interstitial chemotherapy using thermosensitive gel-coated ricin in nude mice bearing a human hepatoma.

AIM: To investigate the anti-tumor effects and mechanisms of interstitial chemotherapy using intra-tumor injection of thermosensitive gel-coated ricin in nude mice bearing a human hepatoma. METHODS: In a subcutaneous mouse model of hepatoma, saline, blank gel, ricin, or thermosensitive gel-coated ricin (TGR) was injected directly into tumors. Fourteen days later, eight mice in each group were sacrificed. The tumors were removed and weighed for calculating tumor growth inhibition rate. Serum alpha-fetoprotein levels, as well as hepatic and renal functions, were measured. Tumor tissue was analyzed under an optical microscope. Terminal deoxynucleotidyl transferase mediated dUTP nick end labeling was used to detect the apoptotic index. Moreover, caspase-3 activity and protein expression in tumor tissue were examined. The survival time of the tumor bearing mice was determined. RESULTS: Following interstitial chemotherapy by intra-tumor injection of TGR in nude mice, serum alpha-fetoprotein levels were significantly reduced with no significant impact on hepatic or renal functions. The rate of tumor growth inhibition was 58.5% following a single, local injection. Histological analysis revealed abundant necrosis. The apoptotic index was 45.96 +/- 7.41%. Caspase-3 activity was increased, and caspase-3 protein was significantly activated in tumor cells. Compared to the saline group, the survival time of mice in the TGR group was significantly extended. At the observation terminal time, day 120, two mice were still alive and fully recovered. CONCLUSION: Interstitial chemotherapy by intra-tumor injection of TGR was highly efficient and safe for the treatment of nude mice bearing a human hepatoma. Interstitial chemotherapy exhibits inhibitory effects by inducing apoptosis and directly killing tumor cells.

US-guided interstitial chemotherapy using paclitaxel temperature-responsive gel for breast cancer treatment in rat.

INTRODUCTION: This study aims to investigate the therapeutic effect of paclitaxel temperature-responsive gel (PTRG) for interstitial chemotherapy on breast cancer, and to explore a new minimally invasive treatment for breast cancer. MATERIALS AND METHODS: Breast cancer models were induced in rats using subcutaneous transplantation of tumor cells. The rats were then divided into control, paclitaxel injection, gel injection and paclitaxel-gel (PG) group. Following treatment, all animals were checked regularly by ultrasonography to observe changes in the tumors. Biopsy tumor tissues were processed for histopathological examination, and apoptotic index was determined by the terminal deoxynucleotidyl transferase dUTP nick end labeling method. In addition, blood cell count and liver transaminase activity were monitored, and the survival time of rats with cancer recorded. RESULTS: Rats in PG group exhibited liquefaction necrosis of tumors. Ninety days after the experiment, four rats exhibited complete extinction of tumors, indicating full recovery. Pathological examination revealed that the tumor tissues in these rats were mostly necrotic, and the apoptotic index of tumor cells increased markedly compared to PI group. Also, the red blood cell, hemoglobin and white blood cell levels declined significantly in the PI group compared with PG group, while glutamic-pyruvic transaminase and glutamic-oxalacetic transaminase activities significantly increased. Meanwhile, no toxicity due to treatment was observed in PG group. CONCLUSION: Interstitial chemotherapy mediated by PTRG appeared to be a safe and effective treatment for breast cancer in rats. It might have clinical applications for treating human breast cancer.

[Effects of intratumoral injection of microspheres containing cobra venom cytotoxin on transplanted human hepatoma in nude mice].

OBJECTIVE: To evaluate the safety and efficacy of intratumoral injection of polylactic-co-glycolic acid (PLGA) microspheres containing cobra venom cytotoxin in nude mice with transplanted human hepatoma. METHODS: Cytotoxic activity of cytotoxin from cobra venom was determined by using methyl thiazolyl tetrazolium method in vitro. Microspheres containing cobra venom cytotoxin were prepared with a double emulsion-solvent evaporation method. Forty BALB/c nude mice were inoculated subcutaneously in right flank with hepatoma BEL-7404 cells. Thirty-two mice whose tumor size reached about 1.0 cm in diameter, were randomly assigned into normal saline group, blank microsphers group, cytotoxin group and cytotoxin-PLGA group. Nude mice were intratumorally injected with normal saline, blank microspheres, cytotoxin or cytotoxin-PLGA microspheres respectively. Internal echo characteristics and blood flow of tumors were observed by high-frequency ultrasound every week after treatment. Twenty-six days after treatment, the tumors were removed to calculate the inhibition rate of tumor growth. The tumor, heart, liver and kidney tissues were obtained for histopathological examination. RESULTS: The cytotoxin separated and purified from crude cobra venom caused intense cytotoxic effects to the BEL-7404 cells in vitro. The diameter of PLGA microspheres containing cobra venom cytotoxin was about (34.45+/-9.85)microm. Encapsulation rate was up to (78.13+/-8.92)%, and cumulative amount of cobra venom cytotoxin released from the PLGA microspheres in vitro during 30 days was up to 84.3%. After intratumoral injection, tumor volumes and weights in the cytotoxin-PLGA group were lower than those in the normal saline group, with a tumor growth inhibition rate of 52.36%. Observed under a light microscope, most tumor tissues were necrotic. No obvious morphological change could be seen on the liver, kidney and heart tissues. CONCLUSION: The above findings indicate that intratumoral injection of cytotoxin-PLGA microspheres has strong antitumor effect and can obviously lessen systemic toxicity, which may provide an effective and feasible method for hepatocellular carcinoma treatment.

Interstitial chemotherapy with ricin-loaded thermosensitive hydrogel in pancreatic cancer xenograft.

BACKGROUND: Pancreatic cancer is one of the most aggressive malignancies, and has a poor prognosis. Despite efforts made in multiple fields, there has been little success in improving the disease-free survival rate of patients. This study was undertaken to investigate the effectiveness and feasibility of using intra-tumoral injection of ricin-loaded thermosensitive hydrogel for treatment of pancreatic cancer xenografts, attempting to develop a new treatment for human pancreatic cancer. METHODS: BALB/c-(nu/nu) nude mice were inoculated subcutaneously in the right flank with the human pancreatic cancer cells, SW1990. Fourteen days after inoculation, 32 mice, bearing tumors of volume 1.5-2.0 cm3, were randomly assigned to one of four groups, and given an intra-tumoral injection of: (1) saline; (2) 23% w/w thermosensitive hydrogel alone; (3) ricin, 10 microg/kg; or (4) 10 microg/kg ricin loaded in thermosensitive hydrogel. On day 14 after administration, the tumors were excised to calculate the inhibition rate of tumor growth and perform histopathological examination. Tumor cell apoptosis was detected by flow cytometry, and RT-PCR was performed to evaluate the mRNA expression levels of Bcl2 and Bax. RESULTS: Intra-tumoral injection of ricin-loaded thermosensitive hydrogel resulted in remarkable control of tumor growth. The tumor became necrotic by day 14 after administration. The histological results clearly confirmed that the tumor cells were lysed. The percentage of apoptotic cells detected by flow cytometry was higher in the ricin hydrogel group than in the other groups. Semi-quantitative RT-PCR revealed that the mRNA expression level of Bcl2 was down-regulated whereas Bax was upregulated. CONCLUSIONS: Intra-tumoral injection of ricin-loaded thermosensitive hydrogel may provide an effective approach for interstitial chemotherapy in pancreatic cancer. Inducing apoptosis by downregulating Bcl2 expression and upregulating Bax expression may be a key molecular mechanism.

[Effects of interstitial chemotherapy using thermosensitive gel-coated ricin on hepatoma H22-bearing mice].

OBJECTIVE: To explore the efficacy and feasibility of interstitial chemotherapy using thermosensitive gel-coated ricin in hepatoma H22-bearing mice. METHODS: Ricin was purified by chromatography method. The purified ricin was identified by Western blot assay and the purity was determined by high-performance liquid chromatography. BALB/c mice were inoculated subcutaneously in right flank with hepatoma H22 cells. When the tumor size reached about 1.0 cm in diameter, 40 mice were randomly divided into untreated group, thermosensitive gel group, ricin group and thermosensitive gel-coated ricin group. Mice in each group were administered different agents by percutaneous intratumoral injection, including normal saline, thermosensitive hydrogel, ricin and thermosensitive gel-coated ricin. Fifteen days after treatment, the tumors were removed to calculate inhibition rate of tumor growth. The tumor tissues were made into pathological sections to perform histopathological examination. The ultrastructure of tumor tissue was examined by electron microscope examination as well. Blood was collected to detect the hepatic and renal functions. The caspase-3 activity of tumor tissue was determined by using zymologic method with a spectrophotometer. RESULTS: After intratumoral therapy, tumor weight in the thermosensitive gel-coated ricin group was lower than that in the untreated group, with a tumor growth inhibition rate of 71.31%. No obvious hepatic or renal toxicities were detected after thermosensitive gel-coated ricin treatment. Histopathologic observation of the tumor tissue showed massive necrosis and typical apoptosis phenomena, including chromatin margination and apoptotic body. Meanwhile, thermosensitive gel-coated ricin resulted in a significant increase in the caspase-3 activity as compared with the untreated group and the ricin group (P<0.01, P<0.05). CONCLUSION: The above findings indicate that intratumoral therapy with thermosensitive gel-coated ricin has strong antitumor effect and can obviously lessen systemic toxicity, which may provide an effective and feasible method for hepatocellular carcinoma treatment.

[Alterations of expression of G protein-inositol phosphates pathway-related genes in essential hypertension: experiment with spontaneously hypertensive rats].

OBJECTIVE: To investigate the changes of G protein-inositol phosphates pathway-related genes and evaluate the role of such changes in the pathogenesis of essential hypertension. METHODS: The pressures of the caudal arteries and body weights of 30 spontaneous hypertensive rats (SHRs), 7 two-week-old, 7 4-week-old, 6 six-week-old, 6 eight-week-old, 6 ten-week-old, and 6 twelve-week-old, and 38 normotensive Wistar-Kyoto (WKY) rats, 6 two-week-old, 6 four-week-old, 6 six-week-old, 6 six-week-old, 6 eight-week-old, 7 ten-week-old, and 7 twelve-week-old, were measured. Then the rats were killed and their hearts, aortas, livers, and kidneys were taken out. 294 specimens of total RNA were obtained from the tissues of ventricle of heart, aortic smooth muscle, liver and kidney. RNA array was used to determine the mRNA levels of G proteins G11 and Gq, and phospholipase C-beta (PLCbeta). RESULTS: The systolic blood pressures of the 6, 8, 10, and 12-week-old SHRs were 158 mm Hg +/- 8 mm Hg, 174 mm Hg +/- 4 mm Hg, 198 mm Hg +/- 13 mm Hg, and 217 mm Hg +/- 9 mm Hg respectively, all significantly higher than those of the age-matched WKY rats (109 mm Hg +/- 6 mm Hg, 128 mm Hg +/- 5 mm Hg,142 mm Hg +/- 4 mm Hg, and 141 mm Hg +/- 5 mm Hg respectively, all P <0.01). The cardiosomatic ratios of the 10- and 12-week-old SHRs were both significantly higher than those of the age-matched WKY rats (both P < 0.01). The G11 mRNA levels in the heart tissue of the 4, 6, 8, 10, and 12-week-old SHRs were 1.42 +/- 0.35, 1.87 +/- 0.40, 1.96 +/- 0.24, 2.09 +/- 0.38, and 2.34 +/- 0.45, all significantly higher than those of the age-matched WKY rats (1.05 +/- 0.18, 1.25 +/- 0.37, 1.26 +/- 0.35, 1.45 +/- 0.30, and 1.51 +/- 0.42 respectively, P < 0.05 or P < 0.01). The Gq mRNA levels of the 4, 6, 8, 10, and 12-week-old SHRs were 1.12 +/- 0.21, 1.30 +/- 0.26, 1.45 +/- 0.35, 1.77 +/- 0.42, and 2.05 +/- 0.46, respectively, all significantly higher than those of the age-matched WKY rats (0.88 +/- 0.09, 0.96 +/- 0.10, 1.03 +/- 0.10, 1.21 +/- 0.38, and 1.29 +/- 0.39 respectively, P < 0.05 or P < 0.01). Similar results were found in the G11 and Gq mRNA levels of the aorta and kidney tissues. The levels of PLCbeta expression in the heart and kidney tissues of the 4, 6, 6, 8, 10, and 12-week-old SHRs were all significantly increased (P <0.05 or P <0.01). Galpha, Gq, and PLCbeta were not significantly expressed in the liver. PLCbeta was hardly found in the aorta. CONCLUSION: Increase of the expression of G protein-inositol phosphates pathway-related genes is an important molecular biological mechanism in the pathogenesis and development of essential hypertension.

[Effect of Astragalus membranaceus on baroreflex sensitivity in spontaneously hypertensive rats].

OBJECTIVE: To observe the chronicity decompression effect of Astragalus Membranaceus(AM) and evaluate the effect on baroreflex sensitivity (BRS). METHOD: Nineteen spontaneously hypertensive rats(SHR) were randomly divided into four groups. The AM groups were intraperitoneally administered with AM parenteral solution 0.9 mL, 1.2 mL and 1.8 mL respectively and the control group was not given AM for eight weeks. Then the change of blood pressure was observed successivly. After eight weeks, BRS were also determined. At last, the difference of blood pressure and BRS among the groups were compared. RESULT: Blood pressure in the control group became higher and higher frome the third week to the eighth week, but the other SHR admistered with AM showed no changein blood pressure level. We also found that the BRS in AM group was higher than that in the control group(P < 0.01). CONCLUSION: AM can promote the BRS in SHR.

[Serum levels of soluble Fas soluble Fas ligand and soluble IL-2 receptor in patients with coronary heart disease]

OBJECTIVE: To evaluate the relationship between serum levels of soluble Fas(sFas), soluble Fas li gand(sFasL), soluble IL-2 receptor(sIL-2R) and coronary heart disease (CHD). METHODS: With enzyme-linked immunosorbent assay (ELISA) tests, sFas, sFasL and sIL-2R were measured in the sera from 30 patients with CHD and 26 subjects without CHD as controls. RESTULTS: Mean level of sFas was significantly higher in patients with CHD than in controls [(1 583.41+/-174.46)ng/L compared with (1 374.55+/-142.42)ng/L, P<0.01]. Compared with the controls, the mean level of sIL-2R was significantly higher in patients with CHD [(944.50+/-395.59)ng/L compared with (652.45+/-163.36)ng/L P<0.01]. Moreover, in patients with CHD sFas and sIL-2R were positively correlated (r=0.418 P<0.05). Whereas no such difference was found between both groups in sFasL (P<0.05). CONCLUSION: High levels of serum sFas and sIL-2R were associated with CHD, and elevation of sFas may inhibit apoptosis in activated T cells, leading to coronary events.