Bioeffects of low-energy continuous ultrasound on isolated sarcoma 180 cells.

BACKGROUND: The aim of this study was to investigate the mechanism underlying bioeffects of low-intensity continuous ultrasound on isolated sarcoma 180 (S180) cells and cellular responses to these effects. METHODS: After sonication, several structural and functional parameters were examined to elucidate ultrasound-induced cell damage. RESULTS: Instant disruption of the cell membrane might be caused by acoustic cavitation, producing mechanical and chemical effects that acted simultaneously on S180 cells; this could be reflected by immediate (morphological) changes such as membrane permeability, membrane fluidity, lipid peroxidation and the generation of hydroxyl radicals in culture medium. Our results of the delayed effects also indicated S180 cells were sensitive to ultrasound-induced apoptosis, and the rate of apoptosis rose gradually with a prolonged incubation time. The presence of apoptotic cells was identified by a distinct morphological form characterized by membrane blebbing, cell shrinkage, chromatin condensation and DNA fragmentation. Moreover, delayed cytotoxicity was accompanied by an increase in intracellular reactive oxygen species (ROS) and a decrease in the mitochondrial membrane potential, and the two events presented obviously a negative correlation. CONCLUSION: ROS secondarily generated from damaged mitochondria may play a role in the induction of apoptosis.

[Antitumor effect of polysaccharides from cactus pear fruit in S180-bearing mice].

BACKGROUND & OBJECTIVE: Polysaccharide components of some traditional Chinese medicine have certain antitumor effects and can promote immune responses. Extractions from cactus pear fruit can inhibit the proliferation of cervical cancer, ovary cancer and bladder cancer cells, and suppress the growth of ovarian cancer in mice. This study was to observe the antitumor effect of polysaccharides extracted from cactus pear fruit in S180-bearing mice. METHODS: S180-bearing mice were established and divided into five groups: normal saline (NS) group, cyclophosphamide (CTX) group, high, middle and low dose of polysaccharide groups. Tumor inhibition rates, values of thymus index, spleen index, superoxide dismutase (SOD), maleic dialdehyde (MDA) and nitrogen monoxidum (NO) were recorded. Changes in ultra-structures of tumor cells under transmission electron microscopy were observed. RESULTS: The tumor inhibition rates in CTX group, high, middle and low dose groups were 7.78%, 31.13%%, 49.70%, 61.07%, respectively. The thymus index was significantly higher in middle and high dose groups than in NS group [(2.61+/-0.43) mg x g(-1) and (2.65+/-0.73) mg x g(-1) vs. (2.22+/-0.24) mg x g(-1), P<0.05]. The spleen index of high dose group was higher than that of NS group [(6.45+/-0.97) mg x g(-1) vs. (5.42+/-1.13) mg x g(-1),P<0.05]. SOD of middle and high dose groups [(303.12+/-13.03) U/mL and (310.03+/-18.02) U/mL] were higher than that of NS group [(280.12+/-10.01) U/mL](P<0.05). MDA was lower in low, middle and high dose groups [(6.56+/-0.75) nmol/mL, (6.24+/-1.03) nmol/mL and (5.78+/-0.90) nmol/mL, respectively] than that in NS group [(7.39+/-0.51) nmol/mL] (P<0.05). NO was lower in low, middle and high dose groups [(56.12+/-8.60) micromol/L, (50.12+/-10.05) micromol/L, (48.06+/-8.45) micromol/L respectively] than in NS group [(64.14+/-1.25) micromol/L](P<0.05). Under electron microscopy, polysaccharide or CTX treated tumor cells showed typical morphology of early apoptosis with condensed chromatin at the margins of nuclei, disintegrated nucleolus and vacuoles in the cytoplasm. CONCLUSION: Polysaccharides extracted from cactus pear fruit possess certain antitumor effects, which can induce apoptosis, increase antioxidation and promote immune responses.

Synthesis and activity of Combretastatin A-4 analogues: 1,2,3-thiadiazoles as potent antitumor agents.

A series of 4,5-disubstitute-1,2,3-thiadiazole compounds were designed and synthesized as potent anticancer agents, some of them exhibited excellent in vitro and in vivo inhibitory activity.

Cellular toxic effects of combination of low dose of cisplatin and interstitial injection of 32P glass microspheres on mouse solid tumor S180.

BACKGROUND: Although 32P-glass microspheres (32P-GMS) have been used in internal radiotherapy for malignant tumors, it has been one of the key obstacles to improve the effect of radiotherapy. We investigated the cellular and hypersensitive effect of combined use of low dose of cisplatin and interstitial injection of 32P-GMS on mouse solid tumor S180. METHODS: The mice with solid tumor S180 were randomly divided into four groups (controls, cisplatin therapy, 32P-GMS therapy and combination therapy). The specimens of the mice were sectioned two weeks after treatment and weighed. The death rate of tumor cells and the inhibition rate of tumor were calculated respectively. The cell cycle and apoptosis rate were evaluated with flow-cytometry. The ultrastructural changes of the four groups were observed by a transmission electron microscope. The data were analyzed by the chi-square test. RESULTS: The growth of tumor was slower in the combination therapy group than in the simple therapy groups by macrography. The inhibition rate and the death rate of tumor cells of the combination therapy group were significantly higher than those of the control group and the other two simple therapy groups (P < 0.05). More cell damages were displayed in the combination therapy group than in the other groups under the light and electronic microscope. CONCLUSION: Low-dose cisplatin combined with interstitial injection of 32P glass microspheres could be used as an effective hypersensitive regimen for the internal radiotherapy of mouse solid tumor S180.

Redifferentiation of human hepatoma cell induced by 6-(p-chlorophenyl)-3-[1-(p-chlorophenyl)-5-methyl-1 H-1,2,3-triazol-4-yl]-s-triazolo[3,4-b]-1,3,4-thiadiazole (TDZ).

6-(p-Chlorophenyl)-3-[1-(p-chlorophenyl)-5-methyl-1 H-1,2,3-triazol-4-yl]-s-triazolo[3,4-b]-1,3,4-thiadiazole (TDZ) is a derivative of various substituted s-triazolo[3,4-b]-1,3,4-thiadiazoles, which are associated with diverse pharmacological activities. However, the antitumor activity of TDZ is not well understood. To evaluate its role on tumor cell lines, we have examined the effect of TDZ on two tumor lines: human hepatoma cell (SMMC-7721) in vitro and Sarcoma180 tumor (S180) in vivo. The cytotoxicity of TDZ on human hepatoma cells was assessed using the MTT assay. The inhibition on tumor growth was evaluated by means of trypan blue exclusion test in vitro, and using a Sarcoma180 tumor (S180) animal model in vivo. A scanning electronic microscope was used to discover the morphological changes on cell surface, cell electrophoresis was employed to determine the changes of cell surface negative charges, and alpha-fetoprotein was applied as a biomarker of hepatoma. The effect of TDZ on DNA synthesis was determined by a [3H]-thymidine incorporation assay, and cell cycle distribution by flow cytometry. The IC50 value of TDZ on SMMC-7721 cells was 52.9 microg/ml (48 h). However, TDZ could inhibit the growth of SMMC-7721 cells at concentrations far lower than the IC50 value. Treated with the same low concentrations of TDZ, microvilli on the surface of SMMC-7721 cells decreased obviously, electrophoresis rate of cells reduced from 2.14 microm ms(-1) x V(-1) x cm(-1) of control to 1.54 and 1.56 microm x s(-1) x V-1 x cm(-1), the content of AFP dropped from 205.14 +/- 6.41 ng x mg(-1) Pr to 115.68 +/- 3.47 and 78.57 +/- 2.35 ng mg(-1) Pr, and the DNA replication was inhibited by 26.8% and 45.2%. These results indicated that TDZ may inhibit proliferation of cancer cells by reversing SMMC-7721 cells malignant phenotypic characteristics and inducing redifferentiation. Flow cytometry showed that TDZ-treated cells resulted in a higher proportion of cells in S phase compared with untreated cells, and only when the concentration reached 64 microg/ml, the apoptosis could happen at the rate of 4.2%. Detection of the inhibition of Sarcoma 180 tumor growth in vivo showed that TDZ reduced the tumor weight and 69.08% of the growth was inhibited. TDZ could inhibit the proliferation of tumors in vitro and in vivo; the possible antitumor mechanism might be inducing redifferentiation at a lower dosage on vitro.

[Observation and research on the electroporation of S-180 cell induced by electric pulse].

This is our first time report of S-180 cell handled with various voltages, capacity and pulse number. Many pores on the S-180 cell membrane can be observed under scanning electron microscope. By the stains of trypan blue, we have known the influence of electric parameters on the ratio of poration. The results show that the ratio of electroporation has positive correlation with the voltages and the pulse number while negative correlation with the capacity.

Ultrastructure of sarcoma 180 cells after ultrasound irradiation in the presence of sparfloxacin.

BACKGROUND: Sonodynamic synergistic antitumor effects have been well described to date, but detailed ultrastructural studies in this field are still lacking. MATERIALS AND METHODS: A suspension of sarcoma 180 cells was exposed to ultrasound (US) in the presence of sparfloxacin (SPFX), one of the new quinolone antibiotics, at 2 W (0.64 W/cm2) for 30 seconds. The antitumor effect was evaluated by the survival rate of cells and cell morphology by scanning and transmission electron microscopy. RESULTS: After US irradiation, the survival rate of tumor cells in the SPFX-added group was 30.9%, significantly lower than 78.7% in the control group (p = 0.0002). Most cells in the control group were spherical, but in the SPFX-added group the cells were aspherical. Cell membranes were often ruptured and numerous pores of various sizes were observed. Some cells were totally disintegrated. CONCLUSION: The most characteristic changes of the synergistic antitumor activities of US and SPFX were on the cell membrane.

[Antitumor effects of electrothermal and electrochemical therapy on mice with sarcoma180].

OBJECTIVE: To explore the anti-tumor effects and the mechanism of a new treatment for Sarcoma180 in mice using both hyperthermia (H) and electrochemical therapy (ECT). METHODS: A group of mice with Sarcoma180 (1 cm in diameter) were divided randomly into four groups and given different treatments: Control, ECT alone, H alone and electrothermal & electrochemical therapy (ETECT). The volumes of the tumors were calculated everyday after treatment. The tumor tissues were examined morphologically at 0, 6, 24 and 72 hours after treatment. RESULTS: The tumors treated with ETECT were completely destroyed, and did not recur within a period of ten days by pathological examination. Results of this group were significantly different with the other groups (P<0.01). However, recurred tumors were found in 6 mice of H group and 5 mice of ECT group, respectively. CONCLUSIONS: Based on the results obtained, ETECT induced by ETECD (D-device) is an effective way to eliminate cancer. It is capable of completely destroying cancer cells in a short time. The rate of Sarcoma180 inhibition is 100%, and malignant cells can't recur. The research provided theoretical and experimental bases for the treatment of oral cancer by a new approach to treat oral cancer.

Antitumor, genotoxicity and anticlastogenic activities of polysaccharide from Curcuma zedoaria.

The antitumor effect of the partially purified polysaccharide from Curcuma zedoaria was studied in mice transplanted with sarcoma 180 cells. The polysaccharide fraction, CZ-1-III, at dose of 6.25 mg/kg/d showed 50% inhibition in solid tumor growth. When mice were injected with fractions, CZ-1 and CZ-1-III, at the dose of 100.0 mg/kg, 91.6% and 97.1% of tumor growth were inhibited, respectively, indicating that the cytotoxic effect of polysaccharide on sarcoma 180 cells increases upon increasing the amount of polysaccharide administered. To assess the genotoxicity of CZ-1-III fraction, several classical toxicological tests were performed. In Ames test, CZ-1-III did not show any transformation of revertant with or without S-9 metabolic activating system, indicating the lack of mutagenic effect of the compound. To assess clastogenic effect, micronucleus and chromosomal aberration assays were performed using Chinese hamster lung (CHL) fibroblast cells. However, up to 259.0 microg/ml concentration of CZ-1-III, neither micronucleus formation nor chromosomal aberration was induced regardless of the presence of S-9 metabolic activating system. Inhibition of CZ-1-III on micronucleus formation induced by mitomycin C was exhibited in a dose-dependent manner, maximally up to 52.0%. These results strongly suggest that CZ-1-III, the polysaccharide fraction from C. Zedoaria, decreases tumor size of mouse and prevents chromosomal mutation.

Ultrastructural study of the TG180 murine sarcoma cell invasion by Toxoplasma gondii: comparison between in vivo and in vitro cell cultures.

Infection of non-adherent TG180 murine sarcoma cells with Toxoplasma gondii was compared, at the ultrastructural level, in both in vivo and in vitro conditions. Suspensions of 3.0 x 10(6) TG180 cells infected in vitro with 1.0 x 10(6) parasites of the RH strain were harvested between the first and 6th day post-infection and processed for transmission electron microscopy. In vivo infection was made by intraperitoneal inoculation in mice of 1.0 x 10(6) TG180 cells, that were co-inoculated with a parasite suspension at the same cell concentration. Cells were harvested 10, 20, 30 min and 24, 48 h post-inoculation and processed for transmission electron microscopy at the same conditions of the in vitro culture. It was observed TG180 murine sarcoma cells with intense and equivalent intracellular parasitism in both conditions. Host cells with parasitophorous vacuoles containing up to 16 parasites, as well as parasites undergoing mitoses or presenting a bradyzoite-like morphology, were frequently seen in both culture methods.

Interaction between fibronectin-bearing surfaces and Bacillus Calmette-Guérin (BCG) or gelatin microparticles.

Gelatin, prepared commercially by degradation of animal collagen, was studied to see whether it had an affinity for fibronectin, which has a known affinity for collagen, and whether gelatin-based drugs could be used to target fibronectin-excreting tumours. Bacillus Calmette-Guérin (BCG) vaccine, an attenuated strain of Mycobacterium bovis, is currently the most effective treatment for superficial transitional cell carcinoma of the bladder. The living cells of the BCG vaccine associate with the fibronectin-bearing surfaces of the tumour. Using a multi-well culture plate technique, gelatin microparticles were shown to be adsorbed onto murine S180 sarcoma cells and this reaction was substantially inhibited by the addition of human plasma fibronectin. The avidities of various BCG substrains and gelatin microparticles for glass-bound fibronectin were measured and the association constants determined. The gelatin microparticles associated with the fibronectin with equal avidity as the BCG cells. The results suggest that this model system may allow the investigation of gelatin-based drug delivery devices capable of targeting fibronectin-bearing surfaces associated with some tumours.

[Ultrastructural observation of intratumoral neutrophils and macrophages induced by garlic oil].

After injection of garlic oil in tumor focus a large amount of neutrophils, macrophages and lymphocytes appeared. Some neutrophils and macrophages located adjacent to the tumor cells, some processes of neutrophils and macrophages penetrated into intracellular body of tumor cells. This result showed that garlic oil could induce neutrophils and macrophages against tumor.

Cell volume alterations induced by platinum-thymine in sarcoma-180 ascites cells: ultrastructural study.

Ultrastructural studies of the effects of the chemotherapeutic agent platinum-thymine, on the morphology of sarcoma-180 ascites cells were studied to elucidate the cancer cells immediate response to therapy and the possible mode of cancer cell regression. Sarcoma-180 ascites cells treated with platinum-thymine in vitro at concentrations of 60 micrograms/ml at varying time intervals demonstrated that the drug is capable of killing cancer cells. The cells exhibited drastic nuclear and cytoplasmic alterations with few lipid spherules in the cytoplasm. The cell volume increased tremendously, with the nucleus relatively larger than the cytoplasm as time of treatment increased. Degradative features of the nucleus, and especially the nucleolus, inhibited metabolic processes. Nucleolar and cytoplasmic necrosis with increased cell volume and vacuolation are potential signals for cancer cells response to the drug and a possible mode of causing the eventual demise of the cancer cells.

[Influence of laurocapram on ultrastructures of mouse skin and sarcoma 180 membrane].

Laurocapram (Lau), 1-dodecyl-hexahydro-2 H-azepin-2-one, (azone) is a new percutaneous penetration enhancer. However, the mechanism of its action for absorption promoter of other agents is still unknown. In this paper the effect of Lau on ultrastructures of skin surface and tumor cell membrane were studies. Lau (2%) suspension was applied to abdominal skin of ICR/JCL, C 57 BL mice or one side of abdominal skin of nude mouse with drug and other side with the vehicle solvent once daily for 2-3 d. The skin was excised at 4 h after the final medication for examination under scanning electron microscope (SEM). The results showed the numerous small infolding lines which divided the skin surface into small areas with vesiculation and peeled the epidermal surface to form a few minor holes. The cuticles of the hair shaft dropped off and became thinner. Numerous desquamated cells around the orifice of the hair were fractured, detached and widened. Sarcoma 180 cells were incubated with Lau 25 micrograms/ml at 37 degrees C for 4 h. The microvilli of some cells dropped off and the size of villi became thinner and shorter. The top of some villi of the cells appeared occasionally thick to make the profile as a bat. The surface of numerous naked cells became rugged and rough and showed many black minor holes in the area of denuded cell membrane or dropped microvilli. More than 100 holes in the exposed surface of the naked cell were seen. It seemed that the Lau drilled holes on the biomembrane and enlarged the orifice of hair follicles and thus enhanced the transdermal absorption.

[Tumor-associated macrophages in tumor neovascularization--in situ ultrastructural cytochemical and quantitative study].

Relation of tumor-associated macrophages to tumor neovascularization in murine transplanted tumor, sarcoma 180(S180), was quantitatively analysed in situ on days 3, 6 and 9 after tumor implantation. Infiltrating macrophages in tumor were identified in paraffin section by alpha-naphthyl butyrate esterase stain and subdivided by electron microscopy according to the peroxidase activity. Simultaneously, the structure of newly formed microvessels was quantitated under light microscope by stereological morphometry. The results showed that, as the tumor grew and blood vessels proliferated, the number of infiltrating macrophages in tumor increased obviously but the ratio of peroxidase (PO) positive mononuclear phagocytes tended to decrease in contrast to the increase of PO-negative phagocytes. Microvasculature volume was found to be positively correlated to the proportion of PO-negative macrophages but negatively correlated to the proportion of PO-positive mononuclear phagocytes. It was indicated that tumor angiogenesis was related to the mature macrophages in the tumor.

Intracellular collagen fibrils: evidence of an intracellular source from experiments with tendon fibroblasts and fibroblastic tumour cells.

This study was designed to substantiate one or both of the two hypotheses for the explanation of intracellular collagen fibrils in collagen-producing cells. The more obvious is the phagocytosis of extracellular collagen fibrils by the cell and the other is a form of autophagocytosis of newly synthesised collagenous products. Information was collected on fibroblasts from murine tendons after exercise and simultaneously stimulating collagen synthesis by treatment with an anabolic steroid hormone. Moreover, in vivo and in vitro fibroblastic tumour cells which demonstrate enhanced protein synthesis were also treated with the anabolic steroid. The findings of intracellular collagen fibrils in tendon fibroblasts and the sarcoma cells after experimentally stimulating collagen synthesis are discussed in the light of the hypothesis that the findings may represent steps of autophagocytosis of newly synthesised collagenous products in the absence of a control mechanism to remove collagenous products which cannot be secreted.

Quantitative description of nuclear morphology in assessing resistance of sarcoma 180 to adriamycin.

Resistance to adriamycin generally is explained through changes of cell/drug interactions that possibly reflect structural alterations of intracellular targets. One of the main targets of adriamycin is believed to be nuclear chromatin. In order to recognize chromatin alterations, we studied cell nuclei morphology and chromatin structure by means of digital image analysis. The studies were performed in both adriamycin-sensitive and -resistant Sarcoma 180 cell lines which were cultured under growth-stimulated and nonstimulated conditions. Using specially developed methods, we extracted parameters characterizing geometrical, optical, and structural properties of the cell nuclei from light microscopical images. The latter parameters concerned microscopical appearances of condensed chromatin and were described by features of high-optical-density regions. The results demonstrated that the quantitative criteria applied enabled the discrimination of sensitive and resistant cells. The most important parameters are the nuclear size, number, distribution, and optical density of condensed chromatin regions. In addition, the criteria permit recognition of changes related to differences in the growth conditions of the cells. The data of the image analysis suggest that adriamycin resistance in Sarcoma 180 cells is associated with characteristic patterns of cell nuclear morphology which can be described with a sufficient number of appropriate parameters. The advantages of image analysis are evident when these results are compared with the flow cytometric findings. The conclusion is that structural features of nuclear chromatin provide information essential for the assessment of drug resistance.

Spontaneous cell shedding by tumor cells in monolayer culture.

Sarcoma 180 monolayers spontaneously shed single cells and small multicellular aggregates into the surrounding medium to produce a dual population of floating and substratum-attached cells. Shedding was a motility-associated event that occurred when cells attempted to migrate over one another. It resulted from a combination of cell shape change and active motility, which increased sensitivity to fluid shear dislodgement by reducing a cell's surface area of adhesive contact and increasing strain tension at its adhesive contact points. Shedding occurred at all phases of the cell cycle. Extracellular matrix but not conditioned medium enhanced the floating subpopulation by slowing the kinetics of reattachment to plastic and cellular substrate. Although sarcoma 180 cells are anchorage independent in the sense that they grow readily in single cell suspension, they nevertheless exhibited anchorage modulation of their cell cycle. Short periods in suspension produced a mild G1 accumulation, whereas longer periods of anchorage deprivation led to a mild G2 accumulation which appeared to result from an interference with cytokinesis.

Acriflavine-induced surface changes in three tumor cell types and differential sensitivity to lectins.

Phytohemagglutinin (PHA) and concanavalin A (Con A) were used as probes to detect changes in the cell surface of Dalton's lymphoma, sarcoma-180 and Ehrlich's carcinoma after short in vitro exposure to acriflavine. Dye-treated cells showed enhancement of agglutination both by PHA and Con A, and such enhancement was found to be dependent on the time of exposure and concentration of acriflavine. However, PHA-induced percent agglutination seemed to be much higher than that of Con A among the 3 cell types. There were also marked differences among the 3 cell types in order of their sensitivity to lectin-mediated agglutination. The strength of the response was greater in lymphoma to both PHA and Con A than that of sarcoma-180 and carcinoma cells, which appeared to be most resistant. Acriflavine, which is known as an intercalative agent with DNA, induces cell surface changes by promoting lectin-mediated cellular agglutination.