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Objective:To screen small-molecule inhibitors of tyrosine kinase receptor B2 (EphB2) by using a molecular docking method, and to investigate their effect on cutaneous squamous cell carcinoma (CSCC) and possible mechanisms of action.Methods:The three-dimensional structure of EphB2 protein and its ligand binding sites were predicted by using the docking tool Schrodinger, and high-throughput virtual screening of EphB2 inhibitors was carried out by molecular docking. The anti-CSCC effect and mechanism of action of the screened EphB2 inhibitors kaempferitrin and aloe-emodin (AE) were verified in in vitro and in vivo experiments. In the in vitro experiments, human CSCC cell lines A431 and SCL-1, as well as the human immortalized keratinocyte HaCaT, were all divided into blank control group, dimethyl sulfoxide (DMSO) group, AE group and kaempferitrin group. Methyl thiazol tetrazolium (MTT) assay (AE at concentrations of 20, 40, 80, 160 μmol/L, kaempferitrin at concentrations of 12.5, 25, 50, 100 μmol/L), scratch and Transwell assays (AE at a fixed concentration of 80 μmol/L, kaempferitrin at a fixed concentration of 50 μmol/L) were performed to analyze the effect of EphB2 inhibitors on the proliferation, migration and invasion of CSCC cells. In the in vivo experiments, specific pathogen-free BALB/c female nude mice were subcutaneously injected with 0.2 ml of A431 cell suspension. After tumor growth, 24 tumor-bearing mice were randomly and equally divided into 4 groups: AE group and kaempferitrin group intraperitoneally injected with 20 mg·kg -1·d -1 AE and 25 mg·kg -1·d -1 kaempferitrin respectively, blank control group and DMSO group intraperitoneally injected with the same volume of sodium chloride physiological solution and DMSO respectively; the tumor size and body weight of nude mice were measured weekly; after consecutive treatment for 28 days, transplanted tumors were resected from the nude mice for hematoxylin and eosin (HE) staining, and real-time fluorescence-based quantitative PCR (qRT-PCR) and Western blot analysis were performed to analyze the effect of AE and kaempferitrin on the mRNA and protein expression of E-cadherin, vimentin, glycogen synthase kinase 3β (GSK-3β), phosphorylated GSK-3β (p-GSK-3β) and β-catenin respectively. One-way analysis of variance and t test were used for comparisons between groups. Results:Two small-molecule compounds AA-504/20999031 (kaempferitrin) and AA-466/21162055 (AE) with high inhibitory activity against EphB2 were screened out. MTT assay showed that both AE and kaempferitrin exhibited strong cytotoxicity to SCL-1 and A431 cells compared with HaCaT cells, and their toxicity increased with the increase of their concentration ( F = 17.95, 11.34, respectively, both P < 0.001) ; after 48-hour treatment, the 50% inhibitory concentrations (IC50s) of AE against SCL-1 and A431 cells were 124.59 and 80.85 μmol/L respectively, and the IC50s of kaempferitrin against SCL-1 and A431 cells were 119.64 and 64.96 μmol/L respectively. Scratch assay showed that the migration distance of A431 cells was significantly shorter in the AE group and kaempferitrin group (36.7 ± 1.0 μm, 44.7 ± 3.5 μm, respectively) than in the DMSO group (88.1 ± 1.4 μm, F = 52.34, P < 0.001), while there was no significant difference in the migration distance of HaCaT cells among the above groups ( F = 1.73, P = 0.238). Transwell assay showed that the number of A431 cells crossing the Transwell membrane significantly decreased in the AE group and kaempferitrin group (145.0 ± 2.5, 94.7 ± 4.1, respectively) compared with the DMSO group (195.3 ± 5.7, F = 72.85, P < 0.001), while neither AE nor kaempferitrin showed significant inhibitory effects of on the number of HaCaT cells crossing the Transwell membrane ( F = 3.91, P = 0.055). The animal experiment revealed significantly decreased volumes of transplanted tumors in nude mice in the AE group and kaempferitrin group (407.42 ± 70.37 mm 3, 368.77 ± 62.7 mm 3, respectively) compared with the DMSO group (841.88 ± 84.63 mm 3, F = 73.78, P < 0.001). HE staining confirmed that AE and kaempferitrin could improve pathological changes of transplanted tumors. qRT-PCR and Western blot analysis showed that AE and kaempferitrin significantly up-regulated the mRNA and protein expression of E-cadherin and p-GSK-3β in tumor tissues (all P < 0.001), and down-regulated the mRNA and protein expression of vimentin, β-catenin and GSK-3β (all P < 0.001) . Conclusion:The small-molecule inhibitors screened by molecular docking can form a stable complex with EphB2, and inhibit the progression of CSCC by affecting the Wnt/β-catenin pathway-induced epithelial-mesenchymal transition.
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Objective:Enriching and isolating breast cancer stem cells from breast cancer transplantation tumors in nude mice.Methods:Human breast cancer MDA-MB-231 cells were injected into the right axilla subcutaneous of 20 nude mice, and the tumor growth was observed .After 30 days, tumors were isolated and stained with hematoxylin and eosin, and then tumor cells from tissues were isolated. DMEM medium containing serum was used to cultivate isolated transplantation tumor cells, cell morphology and growth were also observed. Flow cytometry was used to detect the proportion of stem cells (CD44 +/CD24 -/low cells) in transplantation tumor cells. Serum-free DMEM medium containing epidermal growth factor (EGF), basic fibroblast growth factor (bFGF) and B27 cell supplement were used to cultivate transplantation tumor cells and to obtain cell microspheres. The proportion of stem cells on the 10th day in cell microspheres was detected by using flow cell sorter and stem cells were isolated according to the markers of cell surface. Results:After subcutaneously injecting MDA-MB-231 cells into 20 nude mice for 9 days, 17 nude mice had subcutaneous tumors with more parenchymal cells, little interstitial cells, arranged cords tumor cells, large volume of the cell and abundant cytoplasm, the nuclei in different sizes and hyperchromatic state, mitotic more common, the nucleoli clear and obvious pleomorphy. After cultivating transplantation tumor cells with DMEM medium containing serum, the cells began to grow adherent after 24 h, and the adherent proportion rose to 60% after 3 days; after 7 days, the cell proliferation was accelerated; and the cell morphology was more consistent, most of which were spindle shaped and were not significantly different from MDA-MB-231 cells; the proportion of stem cells in transplantation tumor cells was (0.10±0.02)%. After cultivating transplantation tumor cells with serum-free DMEM medium containing cell cultured supplement, the cells grow in spherical patterns, the proportion of stem cells in cell microspheres got up to (70.47±2.03)% on the 10th day.Conclusions:Subcutaneously injecting MDA-MB-231 cells in nude mice can build breast cancer nude mice ectopic transplantation tumor model. Breast cancer stem cells in the transplantation tumors can be enriched from isolated transplantation tumor cells through serum-free medium, and more stem cells can be isolated to provide the research basis for the biological characteristics of breast cancer stem cells.
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ObjectiveTo investigate the potential mechanism of serum N-glycan alterations in patients with hepatitis B virus (HBV)-related hepatocellular carcinoma (HCC) by measuring serum N-glycan profile and comparing glycosyltransferase gene expression between HCC tissue and adjacent tissue. MethodsThe samples of HCC tissue, adjacent tissue, and normal liver tissue were collected from 34 patients with HBV-related HCC who were admitted to Chinese PLA General Hospital, and serum samples were also collected. Among these 34 patients, 8 were randomly selected and their serum samples were established as HCC experimental group, and the serum samples of 20 healthy adults were established as control group. DNA sequencer-aided fluorophore-assisted carbohydrate electrophoresis was used to analyze serum N-glycan profile in the HCC experimental group and the control group. Quantitative real-time PCR was used to measure the mRNA expression of 8 glycosyltransferase genes (FUT3, FUT4, FUT6, FUT7, FUT8, Gn-TIII, Gn-TIVa, and Gn-TV) in the HCC tissue and adjacent tissue of 34 patients with HBV-related HCC, and Western blot was used to measure the expression of corresponding proteins. The independent samples t-test was used for comparison of continuous data between two groups. ResultsCompared with the control group, the HCC experimental group had a significant increase in the abundance of N-glycan peak9 (NA3Fb) in serum(t=-2.514,P＜0.05). There were significant differences in the mRNA expression of FUT8, Gn-TIVa, and Gn-TV between HCC tissue and adjacent tissue, and the mRNA and protein expression levels of FUT8 and Gn-TV in HCC tissue were significantly higher than those in adjacent tissue (FUT8 mRNA: 1.50±0.34 vs 0.65±0.11, t=-2.354，P=0.022; Gn-TV mRNA: 3.57±0.64 vs 1.33±016, t=-3.384,P=0001; FUT8 protein: 0.70±0.11 vs 0.083±0.017, t=9.555,P=0.001; Gn-TV protein: 1.33±0.19 vs 0.60±0.15, t=5.097,P=0.007). The mRNA expression level of Gn-TIVa in HCC tissue was significantly higher than that in adjacent tissue (2.90±0.47 vs 1.68±0.19, t=-2.403,P=0.019), but there was no significant difference in the protein expression level of Gn-TIVa between HCC tissue and adjacent tissue (052±0.24 vs 0.24±0.11,t=1.833, P=0.141). The changes of glycosyltransferase gene expression in HCC tissue were consistent with the alteration of serum N-glycan profile. ConclusionSerum N-glycan alterations in patients with HBV-related HCC may be closely associated with the upregulated expression of the glycosyltransferase genes FUT8, Gn-TIVa, and Gn-TV in HCC tissue.
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ObjectiveTo investigate the effect of hypoxia-inducible factor-1α (HIF-1α) on the stemness and epirubicin sensitivity of hepatoma cells. MethodsHepatoma cells were selected for experiment. HepG2 hepatoma cells transfected with HIF-1α overexpression plasmid were selected as experimental group, and those transfected with pcDNA3.1 empty plasmid were selected as control group; HepG2 cells alone were selected as HepG2 group. Quantitative real-time PCR was used to measure the mRNA expression of HIF-1α; Western blot was used to measure the protein expression of HIF-1α; flow cytometry was used to measure the expression of CD133 on the surface of hepatoma cells. The three groups of cells were treated with epirubicin at different concentrations (0, 6.25, 12.5, 25, and 50 μmol/L) for 24 hours; MTT assay was used to measure cell viability, and flow cytometry was used to measure apoptosis after treatment with epirubicin (50 μmol/L). A one-way analysis of variance was used for comparison of continuous data between multiple groups, and the t-test was used for further comparison between two groups. ResultsCompared with the HepG2 group and the control group, the experimental group had a significant increase in the mRNA expression of HIF-1α (both P＜0.001), and Western blot showed high expression of HIF-1α in the experimental group. The percentage of CD133 cells was 0.040%±0.003% in the HepG2 group, 0.030%±0.010% in the control group, and 20.110%±0.600% in the experimental group, and the experimental group had a significantly higher positive rate of CD133+ than the HepG2 group and the control group (both P＜0.001). At an epirubicin concentration of 25 and 50 μmol/L, the HepG2 group and the control group had significantly inhibited cell viability and a significantly lower cell viability than the experimental group (both P＜005). After the treatment with 50 μmol/L epirubicin for 48 hours, the experimental group had a significantly lower cell apoptosis rate than the HepG2 group (67.9%±2.5% vs 93.6%±1.5%, P＜0.001) and the control group (67.9%±2.5% vs 93.0%±1.2%, P＜0001). ConclusionHepG2 cells are successfully transfected with HIF-1α overexpression plasmid, and HIF-1α can increase the percentage of liver cancer stem cells and improve their resistance to epirubicin.
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Objective To investigate the effect of atractylone on the viability and apoptosis of hepatoma HepG2 cells and its mechanism of action. Methods Hepatoma HepG2 cells were selected and divided into low-, middle-, and high-dose atractylone groups (5, 10, and 20 μmol/L), and the cells in the control group were added with an equal volume of DMSO. MTT colorimetry was used to measure the viability of HepG2 cells after treatment with different concentrations of atractylone; flow cytometry was used to measure the apoptosis rate and mitochondrial membrane potential of HepG2 cells; the DCFH-DA fluorescent probe labeling method was used to measure the level of reactive oxygen species (ROS) in HepG2 cells; Transwell assay was used to evaluate the effect of atractylone on the migration ability of HepG2 cells; Western blot was used to measure the protein expression levels of Bcl-2, Bax, and cleaved caspase-3. A one-way analysis of variance was used for comparison of continuous data between multiple groups, and the least significant difference t -test was used for comparison between two groups. Results After 24 and 48 hours of treatment with atractylone, compared with the control group, the low-, middle-, and high-dose atractylone groups had a tendency of reduction in cell viability (all P < 0.05), with a half inhibitory concentration of 26.19 μmol/L in atractylone treatment of HepG2 cells for 72 hours. The low-, middle-, and high-dose atractylone groups had a significantly higher apoptosis rate than the control group (14.34%/29.32%/50.12% vs 0.32%, all P < 0.05). Compared with the control group, the low-, middle-, and high-dose atractylone groups had a significant increase in the fluorescence intensity of ROS in HepG2 cells (all P < 0.05). After 48 hours of treatment with atractylone, compared with the control group, the low-, middle-, and high-dose atractylone groups had a significant reduction in the number of migrated cells (132.67±18.36/57.00±9.26/31.00±2.45 vs 258.11±38.54, P < 0.05). Compared with the control group, the low-, middle-, and high-dose atractylone groups had a significant reduction in the expression of the anti-apoptotic factor Bcl-2 and significant increases in the expression of the apoptotic factors Bax and cleaved caspase-3 (all P < 0.05). Conclusion Atractylone can induce the apoptosis and inhibit the migration of HepG2 cells, which provides an experimental basis for further development and utilization of atractylone.
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Objective: To explore the effect of transcatheter embolization combined with radiofrequency ablation (RFA) in treatment of VX2 liver tumors of rabbits. Methods: Sixty rabbit VX2 liver tumor models were randomly divided into 4 groups (each n=15). Fifteen minutes after TACE or transcatheter arterial embolization (TAE), RFA was performed in both TACE+RFA group and TAE+RFA group, respectively.And RFA group was only treated with RFA, TACE group underwent only TACE. Serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) were measured 1 day before and 3 and 7 days after operation. Tumor growth rate, tumor necrosis rate and Suzuki score were measured 7 days after operation. The expression of heat shock protein 70 (HSP70) in liver lesions surrounding ablation zone or embolized zone was detected with immunohistochemistry 1, 3 and 7 days after operation, and hepatocyte apoptosis index and proliferation index were calculated. Results: The levels of ALT and AST in TACE+RFA group were higher than those in other groups 3 and 7 days after operation (all P<0.05). Seven days after operation, the Suzuki score in TACE+RFA group was higher than that in other groups, and tumor growth rates in TACE+RFA and TAE+RFA groups significantly decreased, while the necrosis rates in TACE+RFA and TAE+RFA groups increased compared with RFA and TACE groups (all P<0.05). The expression of HSP70 in hepatic lesions surrounding the ablation zone or embolized zone increased gradually 1, 3 and 7 days after operation, and in TACE+RFA group were higher than in other groups, in TAE+RFA group were higher than in TACE group and RFA group 3 days after operation (all P<0.05). The hepatocyte apoptosis index in hepatic lesions surrounding the ablation zone or embolized zone decreased gradually in all 4 groups 1, 3 and 7 days after operation, while in TACE+RFA group were higher than the other 3 groups, in TACE group 1 and 3 days after operation were higher than in TAE+RFA group and RFA group (all P<0.05). The hepatocyte proliferation index in 4 groups were higher 3 days after operation than that 1 and 7 days after operation, and compared with other groups, in TAE+RFA group were higher 1, 3 and 7 days after operation, in RFA group was higher 1 and 3 days after operation than in TACE+RFA group and TACE group (all P<0.05). Conclusion: The effects of TACE+RFA and TAE+RFA on inhibiting tumor growth are better than TACE and RFA alone for treatment of VX2 liver tumors of rabbits. Compared with TACE+RFA, TAE+RFA can promote hepatocyte proliferation and inhibit hepatocyte apoptosis more effectively with less liver damage.
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Objective To study pharmacokinetics and tissue distribution of CalliSpheres Beads (CB) loaded Arsenic trioxide (ATO) on rabbit VX2 liver tumor by transcatheter arterial chemoembolization (TACE). Method Sixty four rabbits with VX2 liver tumors were randomly divided into 4 groups: control group, CB group, CBATO group and cTACE group. Blood samples were taken at specific time points after TACE.The blood concentration of ATO,liver and kidney functions were examined respectively. In each group, every 4 rabbits were sacrificed on 1 days,3 days,7 days and 14 days after operation. The tumor,liver,kidney, lung,heart and muscle were taken to detect the drug concentration. Bilateral t?test was used to compare the drug concentration in blood and tissue between CBATO group and cTACE group. Results Statistically,The levels of ALT and AST in group CBATO and cTACE on 1st,3rd and 7th days after TACE were significantly higher than those in CB group(ALT: F=25.872, 17.69, 7.016, AST: F=46.365, 32.385, 12.548, P<0.05) respectively. The ALT and AST levels in CBATO group were statistically lower than those in cTACE group (ALT: t=0.369, 0.432, 0.169, 0.353, AST: t=0.488, 0.593, P>0.05). There were no statistically significant differences in the levels of BUN and Scr between the four experimental groups at each observation time point. Statistically, 10 minutes and 20 minutes after TACE, the blood drug concentration inCBATO was significantly lower than that in cTACE (t=7.675, 6.461, P<0.001). while 12 hours after operation,blood drug concentration in CBATO group was higher than that in cTACE group. In tumor tissue,the concentration of ATO in CBATO was higher than that in cTACE,and there was no statistical differences on the 1st day after TACE(t=2.155, P=0.068), but there was a statistical differences between 3rd, 7th and 14th days (t=11.462, 7.624, 2.649, P<0.05). Conclusion CBATO could prolong the time of drug metabolism,increase the drug concentration in tumor tissue,and didn′t aggravate the damage of liver and kidney function.
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Objective To determine whether low frequency ultrasound mediated microbubbles destruction (UMMD) could inhibit VX2 orthotopic hepatic tumor growth in rabbit models.Methods Twenty-four New Zealand white rabbits were implanted with VX2 tumor in left hepatic lobe to establish a homograft rabbit model of liver neoplasms in situ,which were randomly divided into four groups(6 rabbits in each group):group A (intravenous saline only),group B (intravenous microbubbles only),group C (intravenous saline+ low frequency focused ultrasound exposure),and group D (intravenous microbubbles+low frequency focused ultrasound exposure).After 3 days consecutive treatment,tumor volume(TV),and peak intensity (PI) were monitored by conventional ultrasound and contrast enhanced ultrasound(CEUS) on 0,1,7,14 and 21 days after treatment.The rabbits were euthanized at the end of the experiment.Tumor tissues were evaluated by HE stain.Results The parameters of TV and PI of each tumor had no significant difference among four groups before treatment(all P>0.05).TV had no significant difference among four groups on 1 day after treatment(all P>0.05);PI in group C and group D were significantly lower than those in group A and group B (all P0.05).The pathological changes of necrosis tissue,hemorrhagic damage of microvessel and thrombosis were observed in the tumors of group D only,whereas these changes occurred rarely in other groups.Conclusions UMMD can inhibit the growth of VX2 hepatic tumors in rabbits,and be used as a promising novel therapeutic strategy to liver neoplasms.
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The incidence rate of liver cancer tends to increase in recent years, and the molecular and pathogenic mechanisms of liver cancer should be further investigated to guarantee health. As an important model organism, zebrafish have highly conserved genes and grow fast. The early embryo of zebrafish is transparent, which helps with the real-time observation of the development process. Zebrafish are similar to humans in the composition, function, and signaling pathways of hepatocytes, as well as response to injury. In modern biological studies, zebrafish have been wildly used as the model of liver diseases. This article summarizes the research advances in the application of zebrafish as the model of liver cancer, and points out that the techniques for establishing the zebrafish model of liver cancer have become mature. With the constant development of experimental techniques, great achievements will be achieved in the field of liver cancer.
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Objective To investigate the optimal ultrasound exposure parameter on H22 neoplasms of mice meditated by ultrasound exposure combined with self‐made nanobubbles ,and then observe their therapeutic effect combined with cisplatin and their possible mechanism of anti‐tumor . Methods Thirty mice engrafts models with subcutaneous H22 neoplasms were established and divided into 6 groups randomly ,which received ultrasound exposure at different intensity and exposure time . The contrast enhanced ultrasound imaging ( CEUS ) was performed in every group at the four time points of before treatment and at 0 h ,24 h ,72 h after treatment . To obtain the optimal ultrasound parameters ,the tumor inhibitory effect was assessed by enhanced intensity ( EI) and microvascular density ( MVD) . The H22 tumor were treated by ultrasound exposure nanobubbles combined with cisplatin to observe their tumor growth inhibition rate ,and the microvessels density and nuclear associated antigen Ki‐67 proliferation index were measured by immunohistochemical staining . Results There was a statistically difference in enhanced intensity (EI) between the experimental groups and control group ( P < 0 .05) . With the increasing of ultrasound intensity and exposure time ,the tumor inhibitory effect was more obvious ,with an increasing side reactions . Except the simple ultrasound group ,there was a statistical difference in tumor inhibition ,the mean MVD and the tumor cell proliferation index (KI‐67) between control group and the other ultrasound therapy groups ( P<0 .05) . The tumor inhibitory rate was the highest ( tumor inhibition rate 70 .0% ) and the mean MVD and KI‐67 expression were the lowest ( P <0 .05) in the combination group comparing with the others . Conclusions The ideal ultrasound exposure parameter of tumor inhibition showed that exposure intensity chose 1 W/cm2 and exposure time chose 1 min or 3 min intermittence . The ultrasound exposure self‐made nanobubbles combined with cisplatin could enhance the tumor inhibitory effect .Its mechanism may be related to the decrease of microvascular density ,the inbition of tumor cell proliferation and the increase of tumor cell necrosis .
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Objective To investigate the expression of Jagged1 in human epithelial ovarian carcinoma tissues and the effect of Jagged1 on growth of xenograft in nude mice. Methods (1) Forty-eight cases of ovarian cancer and 30 cases of patients with benign epithelial ovarian tumor in the Henan Province Xinxiang Central Hospital during Feb. 2011 to Mar. 2014 were enrolled in this study. The mRNA expression of Jagged1, Notch1 and the downstream target genes Hes1, Hey1 were analyzed by using realtime PCR method. (2) The ovarian cancer xenograft models in nude mice were constructed by injecting SKOV3 cells in axillary subcutaneouswere. The nude mice were randomly divided into Jagged1 interference group, blank plasmid group and control group. Each group had 10 mice. They were transfected with pcDNA3.1(+)-siRNA-Jagged1, blank plasmid pDC3.1 and phosphate buffer, respectively. The tumor volumes and tumor masses were measured 14 days after transfection and the inhibition rate was calculated. The relative mRNA expression of Jagged1, Notch1, Hes1 and Hey1 in xenograft tissues after transfection in each group was detected by using realtime PCR technique and the relative protein expression of Jagged1, Notch1, Hes1 and Hey1 in xenograft tissues was detected by utilizing western blot method. Results (1) The relative mRNA expression of Jagged1, Notch1, Hes1 and Hey1 in ovarian cancer tissues were higher than benign ovarian tumor tissues, the differences were statistically significant (P0.05). Conclusions Jagged1 is highly expressed in epithelial ovarian carcinoma. Jagged1 gene interference in xenograft tumor can inhibit ovarian cancer cell growth and improve tumor suppressor rate, which probably play roles by inhibiting Notch1 signaling pathway.
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Objective To investigate the effect of elemene on mRNA expressions of tumor necrosis factor (TNF) receptor associated factor 6 (TRAF6) and caspase-8 in tumor tissues of mice bearing hepatoma H22.Methods Forty BALB/c mice models bearing hepatoma H22 were established by subcutaneous inoculating tumor cells.Forty BALB/c mice were randomly divided into 4 groups:model group,low-and high-elemene dosage groups,and cisplatin group.The tumors after executing mice were weighted.The mRNA expressions of TRAF6 and caspase-8 in tumor tissues were detected by quantitative real-time reversetranscription polymerase chain reaction (RT-PCR).Results The dosage of elemene could inhibit tumor growth.The inhibition ratio of cancer in the low-and high-elemene dosage and cisplatin group was 24.2％,27.4％,and 28.2％,respectively.It reduced significantly tumor weights(P ＜0.01).Compared to the model group,the expression level of TRAF6 mRNA on tumors was decreased significantly,while the expression level of caspase-8 mRNA was increased significantly in the other groups(P ＜ 0.05).Conclusions The present results indicated that molecular mechanism of inhibition of liver cancer growth treated by elemene might be through down-regulating mRNA expressions of TRAF6 and caspase-8,promoting tumor cells apoptosis,and achieving the anti-tumor effect.
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Objectives To explore the effects of the blood supply extent on the temperature of target site of tumor and volume of coagulation necrosis (V),and to provide experimental evidence for further precise control of dosage of high intensity focused ultrasound (HIFU) and improve the efficiency of HIFU in the treatment.Methods Twenty-four rabbit liver VX2 tumor models were established and were divided into four groups:10 d group,15 d group,20 d group,no blood supply group (rabbits were put to death in the 10 d after the models were established).The same irradiation parameters of HIFU were used to irradiate the hepatic tumor tissue of every group,the real-time temperature of target site of tumor were measured,the software from temperature controller was used to plot the time-temperature curve (TTC),V was measured after HIFU.Residual tumor tissue was resected for pathological observation and microvascular density (MVD) determination.Results (1) Histopathological analysis showed that the extent of a tumor's blood supply followed the order 10 d group ＞ 15 d group ＞ 20 d group (P ＜ 0.01).(2)Tmax,T1,k1:betweengroups had no statistical difference among 10 d group,15 d group,20 d group (P ＞0.05),T T1,and k1 between three groups with no blood supply group had statistical difference (P ＜ 0.05).(3) k2:10 d group ＞ 15d group ＞ 20d group ＞ no blood supply group;T2:10d group ＜ 15d group ＜ 20d group ＜ no blood supply group;(4)V:10 d group ＜ 15 d group ＜ 20 d group ＜ no blood supply group.Conclusions The extent of a tumor's blood supply had a significant effect on the temperature-decrease phase but not on the temperature-increase phase during HIFU treatment.The more abundant blood supply of the tumor was,the faster heat abstraction after HIFU was;and the easier the tissue return to normal temperature,the smaller the volume of coagulation necrosis tissue were.
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Objective To investigate the production and mechanism of chemokine (C-C motif) ligand 5 (CCL5) by macrophages in U14 cervical cancer-bearing mice during infection. Methods The U14 cervical cancer cells were injected in C57BL/6 mice to induce tumor-bearing condition. Lipopolysaccharide (LPS) was injected into C57BL/6 mice to induce infection. The protein expression of CCL5 in the serum and the CCL5 mRNA expression in inflammatory cells were measured by ELISA and fluorescence quantitative-PCR in four groups. Macrophages were induced in the tumor conditioned medium (TCM) which extracted from mice serum. The protein expression levels of CCL5, prostaglandin E2 (PGE2) and cyclic adenosine monophosphate (cAMP) in the medium and CCL5, PGE2 and cAMP mRNA expression in the macrophages were detected in different groups. In order to determine whether the inhibition was related to PGE2, selective cyclooxygenase 2(COX-2) inhibitor NS398 was used to reverse this phenomenon and protein kinase A (PKA) inhibitor H89 demonstrated the mechanism through blocking cAMP/PKA signaling pathway. Results (1) The protein and mRNA level of CCL5 in tumor-bearing mice were respectively (151±35) pg/ml and 1.0, which were lower than those in the tumor-free mice (691 ± 85) pg/ml and 4.5 ± 0.8, there were significant difference between them (all P<0.05). The protein and mRNA level of PGE2 in tumor-bearing mice were (1 198±83) pg/ml and 5.8±0.8, which were higher than those in the tumor-free mice (187±25) pg/ml and 1.0, the difference were significant (all P<0.05). The protein and mRNA level of CCL5 in tumor-free+LPS mice were (4 049±141) pg/ml and 31.5±2.0, which were higher than those in the tumor-bearing+LPS mice (1 951±71) pg/ml and 12.1±2.8, the difference were also significant (P<0.05). The protein and mRNA level of PGE2 in tumor-free+LPS mice were (676±70) pg/ml and 3.4±0.4, which were lower than those in tumor-bearing+LPS mice (2 550±382) pg/ml and 11.6±0.9, the difference were also significant (all P<0.05). (2) Macrophages were cultured in vitro using TCM derived from mice. The protein and mRNA level of CCL5 in tumor-bearing mice TCM were respectively (1 626 ± 177) pg/ml and 28.6 ± 1.2, which were higher than those in the tumor-free mice TCM [(27 ± 3) pg/ml and 1.0], there were significant difference (P<0.05). The protein and mRNA level of PGE2 in tumor-bearing mice TCM were (790 ± 156) pg/ml and 1.7 ± 0.3, which were higher than those in the tumor-free mice TCM [(448 ± 115) pg/ml, 1.0], the difference were significant (all P<0.05). The protein and mRNA level of cAMP in tumor-bearing mice TCM were (164 ± 30) pg/ml and 1.6 ± 0.3, which weres higher than those in the tumor-free mice TCM [(118 ± 25) pg/ml,1.0], the difference were significant (all P<0.05). The protein and mRNA level of CCL5 in tumor-free + LPS mice TCM were (10 475 ± 742) pg/ml and 212.0 ± 5.7, which were higher than those in the tumor-bearing+LPS mice TCM [(6 375±530) pg/ml, 142.3±2.5], the difference were significant (all P<0.05). The protein and mRNA level of PGE2 in tumor-free+LPS mice TCM were (2 438±95) pg/ml and 4.3±0.7, which weres lower than those in the tumor-bearing + LPS mice TCM [(3 441 ± 163) pg/ml, 5.9 ± 0.3], the difference were significant (all P<0.05). The protein and mRNA level of cAMP in tumor-free+LPS mice TCM were (340 ± 13) pg/ml and 4.1 ± 0.4, which were lower than those in the tumor-bearing + LPS mice TCM [(542 ± 42) pg/ml, 5.4 ± 0.5], the difference were significant (all P<0.05). (3) Using COX-2 inhibitor NS398 in the tumor-bearing+LPS mice, the protein and mRNA level of CCL5, PGE2 and cAMP were (7 691±269) pg/ml and 159.0±8.9, (2 820±152) pg/ml and 4.9 ± 0.3, (465 ± 8) pg/ml and 4.3 ± 0.4, respectively, and there were significant difference (all P<0.05), compared to before treatment. Using PKA inhibitor H89 in the tumor-bearing+LPS mice, the protein and mRNA level of CCL5, PGE2 and cAMP were (8 375±520) pg/ml and 177.0±8.8, (2 650±35) pg/ml and 4.7 ± 0.4, (368 ± 13) pg/ml and 3.1 ± 0.7, respectively, and there were significant difference (all P<0.05), compared to before treatment. Conclusion TCM of U14 cells activated macrophages to release PGE2 could inhibit the expression of CCL5 levels by cAMP/PKA signaling pathway.
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The mouse models of nude mice bearing subcutaneous xenografts derived from tumor cell lines provide a preclinical testing system for exploring novel anticancer therapies. However, due to the lack of heterogeneity and an ability of spontaneous distant metastasis, these traditional models are difficult to accurately simulate the clinical condition of the patient. Recently, some researchers have developed better preclinical models by using patient-derived xenografts (PDXs) in mice. It has been shown that transplanting a variety of patient-derived tumor tissues into an appropriate anatomical site of immunocompromised or transgenic mice can authentically mimic the primary tumor in patients, especially with distant metastatic ability. PDXs can not only faithfully preserve the molecular phenotypes and genomic alterations of the primary tumors in patients, but also reproduce the heterogeneity of primary tumors; therefore, PDXs have been used in investigation of mechanism of drug resistance. This review summarizes the methodology of establishing PDXs, verification of similarity in original tumor and the corresponding xenograft, and the value and limitations of PDXs used in the development of new drugs. Copyright © 2014 by TUMOR.
ABSTRACT
Purpose To establish rabbit VX2 tumor model and to explore the relation between perfusion parameters and the expression of the VEGF in the portal vein VX2 implanting tumor emboli. Materials and Methods VX2 tumor was implanted in the portal vein of eight experimental rabbits. Multi-slice CT (MSCT) perfusion scan was performed after tumor formation to measure and compare portal vein tumor thrombus, hepatic blood lfow (HBF) near tumor foci and far away from tumor foci, hepatic blood volume (HBV), probability of surface area product (PS) and mean transit time (MTT). The VX2 tumor emboli were then resected to analyze the relationship between the liver perfusion parameters and VEGF expression using immunohistochemical method. Results MSCT liver perfusion parameters were not statistically signiifcant between foci close to or far away from the tumor (P>0.05). The HBF, HBV and PS within the tumor emboli were higher than that in hepatic parenchyma (P<0.05) and the MTT was higher (P<0.05). There was positive correlation (r=0.711, 0.646 and 0.626, P<0.05) between the HBF, HBV and PS of portal vein VX2 tumor emboli and VEGF expression, and there was negative correlation between MTT and VEGF expression (r=-0.565, P<0.05). Conclusion MSCT perfusion parameters in the portal vein VX2 implanting tumor emboli and the expression of VEGF are positively related. MSCT can evaluate the angiogenesis of portal vein VX2 implanting tumor emboli.
ABSTRACT
Tumor dormancy is a state that tumor cells long-term exist in human body,without obvious growth. Dormant cell is one of the origins of tumor recurrence or metastasis. The exact mechanism of tumor dor-mancy is still unclear. Combined with cellular and molecular biological techniques,tumor cell transplantation and culture in vitro can establish experimental tumor dormancy-recurrence models. These in vivo and in vitro models can be used to investigate mechanisms of tumor dormancy and to explore novel treatments.
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Objective To investigate the tumor chemical ablation and analgesic effects of compound lauromacrogol,and to explore a new method for ultrasound-guided tumor ablation.Methods 18 VX2 tumorbearing rabbits were randomly divided into saline group,ethanol group and compound lauromacrogol group,and the medicine was intratumoral injected guided by ultrasonography.After 4 times treatment,tumor contrast-enhanced ultrasonography,tumor growth inhibition rate and tumor tissue pathology were performed to evaluate the antitumor effects.The analgesic effect was evaluated using the pain model induced by formaldehyde test.Results Ultrasound-guided intratumoral injection of compound lauromacrogol showed significant antitumor effects with a tumor growth inhibition rate of 63.3 ％,which was higher than that of ethanol group.No apparent enhancement was found under contrast-enhanced ultrasonography,and pathology results confirmed wide necrosis.In saline,ethanol and compound lauromacrogol groups,the average accumulated points were 62.25 ± 9.79,21.00 ± 9.13 and 9.87 ± 3.10,respectively by formaldehyde test.Conclusions Ultrasound-guided chemical ablation using compound lauromacrogol showed complete antitumor and strong analgesic effects,which would be a new method for tumor ablation.
ABSTRACT
Objective To comparative analysis the rabbit VX2 tumors angiogenesis in fatty liver and normal liver,and investigate the correlation of contrast-enhanced ultrasound (CEUS) parameters and their angiogenesis.Methods Rabbit models of fatty liver and normal liver with implanted VX2 tumors were established.Two groups of hepatic backgrounds and VX2 tumors were analyzed by QontraXt quantitative software of CEUS.The expression level of tumor microvascular density (MVD) and vascular endothelial growth factor (VEGF) were detected with immunohistochemical techniques and real-time fluorescence quantitative PCR (FQ-PCR).Results ①No significant difference was found in MVD,VEGF protein and gene expression between VX2 tumors in normal liver and fatty liver.The expression of VEGF gene in fatty liver parenchyma were lower than normal liver parenchyma (P ＜0.05).②There was positive correlation between MVD and peak intensity (r =0.494,P ＜0.05; r =0.591,P ＜0.01) both in fatty liver and normal liver VX2 tumors.The expression of VEGF protein were not correlated with TIC parameters (all P ＞ 0.05).③The MVD had positive correlation with expression of VEGF protein in fatty liver and normal liver VX2 tumors (r =0.508,P ＜0.05; r =0.570,P ＜0.01).Conclusions The expression of MVD and VEGF had no significant difference between fatty liver and normal liver VX2 tumors.The peak intensity of VX2 tumors CEUS had positive correlation with MVD both in fatty liver and normal liver,which can indirectly reflect MVD expression level and help to evaluate tumor angiogenesis.
ABSTRACT
Objective: To investigate the effects of TFPI-2 (tissue factor pathway inhibitor 2) on the growth and angiogenesis of subcutaneous tumor xenografts in nude mice established from human liver cancer cells. Methods: The Hep3B cells stably expressing TFPI-2 (Hep3B-TFPI-2 group) and the Hep3B cells transfected with empty vector PCDNA3.1 (Hep3B-V group) or without transfection (Hep3B-P group) were subcutaneously transplanted into nude mice respectively to generate subcutaneous tumor xenografts. The volume of tumor xenograft was measured every three days, and the growth curve of tumor xenograft was drawn when the subcutaneous tumor xenograft was visible. The nude mice were killed three weeks after transplant, the volume of tumor xenograft was measured, and the total RNAs and proteins in tumor xenografts were extracted. The mRNA and protein expressions of TFPI-2 and VEGF (vascular endothelial growth factor) in tumor xenografts were analyzed by RFQ-PCR (real-time fluorescence quantitative PCR) and Western blotting, respectively. The expression of TFPI-2 protein and the MVD (microvessel density) in tumor xenografts were observed by immunohistochemistry. Results: The eventual tumor volume of tumor xenografts in Hep3B-TFPI-2 group was apparently smaller than those in Hep3B-V group and Hep3B-P group (both P < 0.05). The expression of mRNA and abundance of protein of TFPI-2 in Hep3B-TFPI-2 group were significantly higher than those in the other two groups (P < 0.05); while the expression of mRNA and abundance of protein of VEGF in Hep3B-TFPI-2 group were apparently lower than those in the other two groups. Compared with Hep3B-V group and Hep3B-P group, the inhibitory rates of VEGF protein expression in Hep3B-TFPI-2 group were 19.8% and 23.5%, respectively (P < 0.05). The MVD in Hep3B-TFPI-2 group was apparently lower than those in the other two groups (P < 0.05). Conclusion: TFPI-2 can significantly inhibit the growth and angiogenesis of subcutaneous tumor xenografts in nude mice established from hepatocarcinoma Hep3B cells. Copyright © 2013 by TUMOR.