Enhanced effects of TRAIL-endostatin-based double-gene-radiotherapy on suppressing growth, promoting apoptosis and inducing cell cycle arrest in vascular endothelial cells

This study examined the effects of TRAIL-endostatin-based gene-radiotherapy on cellu-lar growth, apoptosis and cell cycle progression in human vascular endothelial cells ECV304 in vitro. The expression of TRAIL and endostatin protein in ECV304 cells was detected by ELISA after the transfection of recombinant plasmid pshuttle-Egr1-shTRAIL-shES and X-ray irradiation. Then MTT assay was used for determining the cellular proliferation, and flow cytometry (FCM) plus Annexin V and propidium iodide (PI) double-staining or PI single-staining were employed for the detection of apoptosis and cell cycle progression. The results showed that expression of TRAIL and endostatin protein exhibited a time- and dose-dependent change in ECV304 cells after pshut-tle-Egr1-shTRAIL-shES transfection in conjunction with irradiation. In the TRAIL-endostatin-based single- or double-gene-radiotherapy, the cell viability declined in a time- and dose-dependent manner, the percentage of cells at G2/M phase and apoptotic rate was increased, and the percentage of cells at G0/G1 phase was lowered as compared with those receiving radiotherapy alone. Moreover, TRAIL-endostatin-based double-gene-radiotherapy demonstrated better effects on growth inhibition, promotion of apoptosis and induction of cell cycle arrest in ECV304 cells than sin-gle-gene-radiotherapy.     

Role of cellular immunity in halothane hepatitis： an in vitro study

Objective： To explore the effect of cellular immunity in halothane hepatitis. Methods： Hepatotoxicity model was established by exposing male Hartley guinea pigs to 1% halothane via inspiration for 4 h each time for 1 or 3 times within a 42-day interval. Then their hepatocytes and lymphocytes were collected and divided into 2 parts for different cultures. Hepatocytes were cultivated with or without 1% halothane for 4 h and lymphocytes were cultivated with or without 12.5 μg/ml trifluoroacetylated guinea pig serum albumin （TFA-GSA）. Then the 2 kinds of hepatocytes were co-cultivated with lymphocytes （1：100） with or without TFA-GSA induction respectively and the supernatant fluid Was taken after 24, 48 and 72 h to determine the concentration of alanine aminotransferase （ALT）. The halothane cultivated hepatocytes were co-cultivated with various proportion of TFA-GSA antigen induced lymphocytes and ALT was determined after 48 h to determine the proper proportion of hepatocytes and lymphocyte. Results： Lymphocytes of 3 times halothane induced guinea pigs caused a significant increase of ALT in hepatocytes with or without halothane induction. But the lymphocytes of 1 time halothane induced guinea pigs only caused a significant increase of ALT in hepatocytes with induction of halothane. The increase of ALT was only seen after 48- and 72-hour co-culture. The proper proportion of hepatocytes and lymphocytes was 1：100 for lymphocytes cytotoxicity. Conclusion： Lymphocytes is sensitized after inhalation of halothane and generates cytotoxicity to hepatocytes. The immune response of lymphocytes to hepatocytes will be enhanced by repeated inhalation of halothane. The cellular immunity may be one of the mechanisms of halothane induced hepatotoxicity.     

Cytotoxicity of Captafol in Mammalian Cells

The cytotoxicity of captafol,a phthalimide-derived fungicied,was evaluated in IB-RS-2 cells.Captafol at 0.12-1.0μg/ml blocks the cell multiplication.This effect is concentrationdependent,only partially reversible and the degree of inhibition increases with time.The synthesis of DNA and RNA is inhibited in parallel by increasing concentrations of the chemical.     

Cytotoxic effects of sodium dodecyl benzene sulfonate on human keratinocytes are not associated with proinflammatory cytokines expression

Background Keratinocytes play a crucial role in the biological function of skin barrier.The relationship between sodium lauryl sulfate （SLS） and keratinocytes has been studied.However,the cytotoxicity and effects of sodium dodecyl benzene sulfonate （SDBS）,a common detergent similar to SLS,on keratinocytes are still not known.This study aimed to investigate the effects of SDBS on cytotoxicity and expression of proinflammatory cytokines in cultured human keratinocytes.Methods This study was carried out using the keratinocytes cell line,HaCaT cells.The cytotoxicity of SDBS on HaCaT cells was evaluated with cell counting kit-8 （CCK-8） and phase-contrast microscopy.After exposure to different concentrations of SDBS,the total RNA of the HaCaT cells was extracted for evaluating the relative mRNA expression of IL-1α,IL-6,IL-8,and TNF-α by qPCR.The supernatants of cells were collected for measuring the levels of IL-6 and IL-8 by enzyme-linked immunosorbent assay （ELISA）.Results SDBS at concentrations of 20 Jg/ml and over showed direct cytotoxicity and induced morphological changes of the HaCaT cells.The mRNA expressions of IL-1a,IL-6,IL-8,and TNF-α in different concentrations of SDBS at different time were comparable with that of controls.SDBS at concentrations of 5,10,and 15 μg/ml had no significant effects on IL-6 and IL-8 excretion from HaCaT cells after 24-hour exposure.Moreover,no significant effects on the IL-6 and IL-8 excretion were found after 10 and 15 μg/ml S DBS stimulations for 6,12,and 24 hours,respectively.Conclusion SDBS at higher concentrations had cytotoxicity on HaCaT cells but had no effects on the mRNA expression of IL-1α,IL-6,IL-8,and TNF-α,that was different from SLS.     

Effects of TNF-α and curcumin on the expression of VEGF in Raji and U937 cells and on angiogenesis in ECV304 cells

Background To better understand the possibilities of antiangiogenic tumor therapy and to assess possible side effects, we investigated the effect of tumour necrosis factor （TNF）-α and curcumin on the expression of vascular endothelial growth factor （VEGF） in U937 and Raji cell lines and their effect on angiogenesis in a human umbilical vein endothelial cell （HUVECs）-derived cell line （ECV304）, and also the relationship between Notchl and VEGF. The aim of this study was to elucidate potential mechanisms controlling tumor neovascularization. Methods VEGF secreted by U937 and Raji cell lines was determined by ELISA. Angiogenesis was tested by network formation of endothelial cells on Matrigel. Levels of VEGF mRNA in U937 and Raji cells and Notchl mRNA levels in EV304 cells were determined by RT-PCR. Results Secretion of VEGF by U937 and Raji cells was increased by TNF-α treatment and suppressed by curcumin （P 〈 0. 01 ）. The mRNA expression of VEGF165 and VEGF121 （containing 165 and 121 amino acid residues, respectively） were detected in any fractions. TNF-α augmented the expression of VEGF165 and VEGF121 mRNA and curcumin reduced the expression （P 〈0. 01 ）. No networks or cords formed in control and curcumin groups. There was tube formation on matrigel in the supernatants of the Raji culture group and the supernatants groups treated by VEGF group and TNF-α in Raji cell. Notch1 mRNA was detected but there was no significant change in the VEGF group compared with control （P 〉 0. 05）. Conclusions Expressions of VEGF mRNA in U937 and Raji cells were increased by TNF-α and suppressed by curcumin. VEGF and TNF-α can induce angiogenesis, and curcumin can inhibit angiogenesis in ECV304 cells.     

Inhibitive effect of cremophor RH40 or tween 80-based self-microemulsiflying drug delivery system on cytochrome P450 3A enzymes in murine hepatocytes

This study examined the effect of self-microemulsiflying drug delivery system (SMEDDS) containing Cremophor RH40 or Tween 80 at various dilutions on cytochrome P450 3A (CYP3A) enzymes in rat hepatocytes, with midazolam serving as a CYP3A substrate.The particle size and zeta potential of microemulsions were evaluated upon dilution with aqueous medium.In vitro release was detected by a dialysis method in reverse.The effects of SMEDDS at different dilutions and surfactants at different concentrations on the metabolism of MDZ were investigated in murine hepatocytes.The cytotoxicity of SMEDDS at different dilutions was measured by LDH release and MTT technique.The effects of SMEDDS on the CYP3A enzymes activity were determined by Western blotting.Our results showed that dilution had less effect on the particle size and zeta potential in the range from 1:25 to 1:500.The MDZ was completely released in 10 h.A significant decrease in the formation of 1’-OH-MDZ in rat hepatocytes was observed after treatment with both SMEDDS at dilutions ranging from 1:50 to 1:250 and Cremophor RH 40 or Tween 80 at concentrations ranging from 0.1% to 1% (w/v), with no cytotoxicity observed.A significant decrease in CYP3A protein expression was observed in cells by Western blotting in the presence of either Cremophor RH40 or Tween 80-based SMEDDS at the dilutions ranging from 1:50 to 1:250.This study suggested that the excipient inhibitor-based formulation is a potential protective platform for decreasing metabolism of sensitive drugs that are CYP3A substrates.     

Effect of TNF-alpha and IFN-alpha on the proliferation and cytotoxicity of lymphokine-activated killer cells in patients with bladder cancer.

OBJECTIVE To investigate the effects of interleukin-2 (IL-2) combined with either tumor necrosis factor-alpha (TNF-alpha) or alpha-interferon (IFN-alpha) on the proliferation and cytolysis to bladder tumor cells of lymphokine-activated killer (LAK) cells in patients with bladder cancer.

METHODS LAK cells were generated by ficoll-paque density-centrifugation from 21 patients with bladder cancer and cultured in medium containing IL-2. LAK cell proliferation was assayed in the presence of various concentrations of either TNF-alpha or IFN-alpha by cell count in 96-well plates. Bladder cancer cell lines BIU-87 and EJ were cultured as target cells and the cytotoxicity of LAK cells was determined by 3-(4,5-dimethylthiazol-2-yl)-2, 5 diphenyltetrazolium bromide (MTT) assay.

RESULTS The proliferation of LAK cells induced by IL-2 was enhanced by TNF-alpha in a dose-responsive fashion. The direct growth support for the LAK cells was also observed with IFN-alpha at the concentration of 1000 U/ml after 48 hours of culture. TNF-alpha (5000 U/ml) resulted in an increase in the cytotoxicity of LAK cells to BIU-87 and EJ cells. However, the change of cytotoxicity of LAK cells treated with IFN-alpha was not statistically significant.

CONCLUSIONS TNF-alpha and IFN-alpha enhance the proliferation and activation of LAK cells and influence their antitumor cytotoxicity in patients with bladder cancer.

     

Effect of polysaccharide sulfate 916 on the production of nitric oxide in ECV3 04 cells induced by cytokines and H2O2

Objective To investigate the effect of polysaccharide sulfate 916 (PS916) on the productio n of nitric oxide (NO) in ECV304 cells induced by tumor necrosis factor-α (TNF α), interleukin-1β (IL-1β) and H(2)O(2) in vitro. Methods Production of NO in ECV304 cells was measured by the Griess method and the proli feration of cells was tested by the MTT method. The activity of NO synthase was detected spectrophotometrically.Results Production of NO in ECV304 cells decreased after treatment with 40 ng/ml IL-1 β and 40 ng/ml TNFα, but increased in the presence of H(2)O(2) 0.1 mmol/L . PS916 significantly enhanced NO production in ECV304 cells in a dose-depende nt manner in the TNFα and IL-1β treated groups and decreased it in the H2O 2 treated group. Proliferation of ECV304 cells was inhibited by TNFα and H 2O2 and no effect was found in the IL-1β treated group. PS916 increased the proliferation of cells treated with TNFα and H(2)O(2) dose-dependently. In vitro, PS916 has no effect on the activity of NO synthase. Conclusion PS916 has a protective effect on ECV304 cells exposed to IL-1β, TNFα and H2 O2.     

Effects of Cypermethrin on Male Reproductive System in Adult Rats

Objective To evaluate effects of cypermethrin on the testis histology and testosterone, LH and FSH in adult male Sprague‐Dawley rats. Methods The intact adult male rats were randomly divided into five groups and were treated with cypermethrin at doses of 0, 7.5, 15, 30, or 60 mg/kg per day by oral gavage for 15‐days. After the treatments, serum was collected for hormone assays. The testes, epididymides, seminal vesicles, and prostates were excised and weighed. The right testis was frozen for daily sperm production and the left one was processed for histopathology. Results Daily sperm production decreased significantly in 30 and 60 mg/(kg day) groups. Testicular structure abnormalities included atrophic and distorted seminiferous tubules, deformed and disordered arrangement of germ cells, reduced germ cells, Sertoli cells and Leydig cells, vacuolization and multinucleated formations of spermatids in the cypermethrin‐treated rats. Vacuolization was found in Sertoli cells and the deformed nucleus was noted in Leydig cells. Serum testosterone reduced significantly in 30 and 60 mg/(kg day) groups. Serum FSH increased significantly in 60 mg/(kg day) group. Conclusion Cypermethrin induces impairments of the seminiferous tubules structure and spermatogenesis in the rats. The damages of the male reproductive system may be attributed to the imbalance of circulating testosterone.     

Low doses of ethanolic extract of Boldo （Peumus boldus） can ameliorate toxicity generated by cisplatin in normal liver cells of mice in vivo and in WRL-68 cells in vitro,but not in cancer cells in vivo or in vitro

OBJECTIVE： Use of cisplatin, a conventional anticancer drug, is restricted because it generates strong hepatotoxicity by accumulating in liver. Therefore its anticancer potential can only be fully exploited if its own toxicity is considerably reduced. Towards this goal, ethanolic extract of the plant, Boldo （Peumus boldus）, known for its antihepatotoxic effects, was used simultaneously with cisplatin, to test its ability to reduce cisplatin＇s cytotoxicity without affecting its anticancer potential. METHODS： The cytotoxicity of Boldo extract （BE） and cisplatin, administered alone and in combination, was determined in three cancer cell lines （A549, HeLa, and HepG2） and in normal liver cells （WRL-68）. Drug-DNA interaction, DNA damage, cell cycle, apoptosis, reactive oxygen species （ROS） and mitochondrial membrane potential （MMP, Aψ） were also studied. Hepatotoxicity and antioxidant activity levels were determined by alanine aminotransferase, aspartate aminotransferase, lactate dehydrogenase and glutathione assays in mice. The cytotoxicity of related proteins was tested by Western blotting. RESULTS： Co-administration of BE and cisplatin increased viability of normal cells, but had no effect on the viability of cancer cells. Boldo protected liver from damage and normalized different antioxidant enzyme levels in vivo and also reduced ROS and re-polarized MMP in vitro. Bax and cytochrome c translocation was reduced with caspase 3 down-regulation. Further, a drug- DNA interaction study revealed that BE reduced cisplatin＇s DNA-binding capacity, resulting in a reduction in DNA damage. CONCLUSION： Results indicated that a low dose of BE could be used beneficially in combination with cisplatin to reduce its toxicity without hampering cisplatin＇s anticancer effect. These findings siqnify a potential future use of BE in cancer therapy.     

Effects of Taohong Siwu Decoction Ⅱ in the Chick Chorioallantoic Membrane （CAM） Assay and on B16 Melanoma in Mice and Endothelial Cells ECV304 Proliferation

To investigate the anti-angiogenesis action of Taohong Siwu Decoction Ⅱ （THSWD Ⅱ）. Methods： The chick chorioallantoic membrane （CAM） assay was adopted to study the anti-angiogenesis action of THSWD Ⅱ; the MTT test was used to investigate its effect on proliferation of the human umbilical vein endothelial cells ECV304; and the immunohistochemical method was used to observe the effect of THSWD Ⅱ on the expression of kinase insert domain containing receptor/fetal liver kinase 1 （KDR/Flk-1） and the microvessel density （MVD） of B 16 melanoma in mice. Results： After treatment with THSWD Ⅱ, the blood vessel index of CAM and the absorbency of ECV304 in the THSWD Ⅱ 1mg/ml group and the 2mg/ml group decreased significantly （P〈0.01）; the weight, the expression of KDR/Flk-1 and the MVD of B16 melanoma in mice reduced significantly in the THSWD Ⅱ 5g/kg group, the 10g/kg group and the TSHSWD10g/kg plus cyclophosphamide group （P〈0.01）. Conclusion： THSWD Ⅱ has the actions of anti-angiogenesis, and inhibiting the proliferation of ECV304 cells and the growth of B16 melanoma. The clinical anti-tumour mechanism is considered to be related possibly to its anti-angiogenesis action by inhibiting the expression of KDR/FIK- 1.     

Effects of glutathione depletion using buthionine sulphoximine on the cytotoxicity in mammalian cells and human tumor cells in vitro.

An inhibitor of glutathione biosynthesis, buthionine sulphoximine (BSO), was used to deplete the endogenous thiols in mammalian cells in vitro. In this study, the cytotoxicity of BSO and BSO combined with the hypoxic cell radiosensitizer misonidazole (MISO) was investigated. Both aerobic and hypoxic cytotoxicity of MISO was found to be increased. The concentration of BSO required to reduce the colony forming ability to 50% (Cc) for the chronic cytotoxicity on V79 cells was 0.03 mmol/L under aerobic condition, while the Cc for the acute cytotoxicity on V79 cells under hypoxic and aerobic conditions was 0.4 and 0.5 mmol/L. The growth inhibition rate of human tumor cells K562 and SGC-7901 by BSO was 6.89-26.06% and 12.01-55.69%, respectively. Enhanced cytotoxicity activity was observed when BSO was used in combination with cis-dichlorodiamino Pt(II) or 5-fluorouracil.

     

Neurotoxic effect of rotenone on dopaminergic neuron PC12 in vitro

Objective： To investigate the mechanism of oxidative stress in rotenone neurotoxicity to dopaminergic neuron PC12. Methods： High differentiated PC12 cells as dopaminergic neurons were treated by different concentrations of rotenone. The morphology was observed with inverted phase contrast microscope and transmission electron microscope. Cell viability and proliferation inhibition were assessed by MTT. SOD and MDA were detected with biochemical assay. And the specific fluorescent probe （DCF-DA） was used to examine ROS in PC12 cells. Results： After treated with rotenone for 24 h, most of the PC12 cells became smaller and rounder. The process of axon was reduced, shortened or broken in a time and concentration dependent manner. The mitochondrial structure and metabolism were changed. Endoplasmic reticulum expanded and the free ribosome increased. Compared with the control group, cell proliferation inhibition increased and cell viability decreased. SOD increased and MDA decreased. The intensity of fluorescence was more obvious in PC12 cells treated by rotenone compared with control group. Conclusion： Rotenone is neurotoxic to cultured dopaminergic neuron PC12. Rotenone might exert this effect through the metabolism of oxidative stress on the pathogenesis of the neuron.     

Combined Effects of 50 Hz Magnetic Field and Magnetic Nanoparticles on the Proliferation and Apoptosis of PC12 Cells

Objective To investigate the bioeffects of extremely low frequency(ELF) magnetic field(MF)(50 Hz, 400 μT) and magnetic nanoparticles(MNPs) via cytotoxicity and apoptosis assays on PC12 cells. Methods MNPs modified by SiO2(MNP-SiO2) were characterized by transmission electron microscopy(TEM), dynamic light scattering and hysteresis loop measurement. PC12 cells were administrated with MNP-SiO2 with or without MF exposure for 48 h. Cytotoxicity and apoptosis were evaluated with MTT assay and annexin V-FITC/PI staining, respectively. The morphology and uptake of MNP-SiO2 were determined by TEM. MF simulation was performed by Ansoft Maxwell based on the finite element method. Results MNP-SiO2 were identified as ~20 nm(diameter) ferromagnetic particles. MNP-SiO2 reduced cell viability in a dose-dependent manner. MF also reduced cell viability with increasing concentrations of MNP-SiO2. MNP-SiO2 alone did not cause apoptosis in PC12 cells; instead, the proportion of apoptotic cells increased significantly under MF exposure and increasing doses of MNP-SiO2. MNP-SiO2 could be ingested and then cause a slight change in cell morphology.Conclusion Combined exposure of MF and MNP-SiO2 resulted in remarkable cytotoxicity and increased apoptosis in PC12 cells. The results suggested that MF exposure could strengthen the MF of MNPs, which may enhance the bioeffects of ELF MF.     

Studies of the Genotoxicity of Glycidyl Methacrylate (GMA)

The following experiments were conducted to evaluate the genotoxic effects of GMA (glycidyl methacrylale) on mammalian and human cells.(1) Using the absorption spectrum shift method in vitro, we observed that the maximums of calf thymus DNA and GMA were shifted toward longer wavelengths (a change of more than 15nm) and the absorbance decreased after incubation at room temperature for 15min or more.The result indicates that binding of DNA and GMA had occurred.The binding force is strong, not affected by the addition of concentrated sodium chloride solution, and only slightly decreased by the addition of 8 M urea solution.Therefore the bond between DNA and GMA might be covalent.(2) In cell cultures, unscheduled DNA synthesis (UDS) in human and/or rat lymphocyte was induced and DNA semiconserva-tive replication was inhibited by GMA at concentrations of less than 5.2 mM.(3) Sperm abnormality tests and assays of UDS in germ cells of male mice were conducted to study the in vivo genotoxicity of GMA.The results revealed that GMA could damage DNA, increase sperm abnormality frequency, and reduce the number of sperm cells, 1990 Academic Press.Inc.     

The expression of TRAIL and its receptors in osteosarcoma cells and the apoptosis effect of a combination of TRAIL, adriamycin and IFN-γ on MG-63 cells

Objective: This study investigated the effects of rh-TRAIL with or without chemotherapeutic drugs on the apoptosis of the osteosa-rcoma cell line, MG-63, and the influence of chemotherapeutic drugs on changes in the expression of DR5 and YinYang1(YY1) in MG-63 cells. Methods: The effects of treatment with rh-TRAIL alone and/or chemotherapeutic drugs on MG-63 cell growth inhibition and apoptosis were measured using the MTT assay, FACS analysis of Annexin V labeled cells, and the mRNA changes of DR5 and YY1 were detected by RT-PCR. Results: Rh-TRAIL protein inhibited the growth of MG-63 cells, and this inhibition was increased by adriamycin and IFN-γ. Adriamycin and IFN-γ significantly facilitated the induction of the expression of DR5 and reduced the expression of YY1. Conclusion: The apoptosis-inducing effect of rh-TRAIL in MG-63 cells was enhanced by chemotherapeutic drugs.     

Expression of CD28 and CTLA4 on T Cells in Bone Morrow of Immune-mediated Aplastic Anemia Mice

Summary： To investigate the expression and significance of CD28 and CTLA4 on T cells in bone marrow of aplastic anemia （AA） mice, in vitro bone marrow mononuclear cells （BMMNCs） were activated through being incubated with PHA （15 μg/mL）. The expression of CD28 and CTLA4 on T cells incubated with or without PHA was detected by two-color flow cytometry. The expression of CD28 and CTLA4 was significantly increased after PHA stimulation. In the AA mice. the expression of CD28 with or without PHA stimulation was both higher than that in the normal mice （both P〈0.01）, but the expression of CTLA4 with or without PHA stimulation showed no significant difference in comparison to that in the normal mice （both P〉0.05）. In the AA mice, there were more activation and activated potential of T cells than the normal, and the abnormal expression of CD28 and CTLA4 may participate in immunological disorder mediated by T cells.