Identification of differentially expressed proteins between human hepatoma and normal liver cell lines by two-dimensional electrophoresis and liquid chromatography-ion trap mass spectrometry

In the previous study, the proteomes of the human hepatoma cell line BEL-7404 and the normal human liver cell line L-02 were separated by high resolution two-dimensional electrophoresis (2-DE). Image analysis revealed that 99 protein spots showed quantitative and qualitative variations that were significant (P < 0.01) and reproducible. Here we report the identification results of some of these protein spots. Protein spots excised from 2-D gels were subjected to in-gel digestion with trypsin, and the resulting peptides were measured by microbore high performance liquid chromatography - ion trap - mass spectrometry (LC-IT-MS) to obtain the tandem mass (MS/MS) spectra. Twelve protein spots were identified with high confidence using SEQUEST with uninterpreted MS/MS raw data. Besides inosine-5'-monophosphate dehydrogenase 2, heat shock 27 kDa protein, calreticulin and calmodulin, whose expression was elevated in hepatoma cells, glutathione-S-transferase P was identified from hepatoma cells in which its level was 18-fold higher compared to human liver cells. Two spots were identified as the homologs of reticulocalbin for the first time in hepatoma cells and their expression increased compared to liver cells. However, tubulin beta-1 chain and natural killer cell enhancing factor B were downregulated in hepatoma cells. A tumor suppressing serpin, maspin precursor, was identified from one spot whose quantity was much higher in the normal liver cell line. More interestingly, epidermal fatty acid-binding protein (E-FABP) and fatty acid-binding protein, adipocyte-type (A-FABP), were detected in liver cells but not in hepatoma cells. The functional implication of the identified proteins was discussed.     

THE EFFECTS OF ULTRAVIOLET C ON in vitro NUCLEOSOME ASSEMBLY AND STABILITY

Abstract The effects of ultraviolet C (UVC) irradiation on nucleosome assembly and its stability were investigated quantitatively using an in vitro nucleosome assembly system comprising a plasmid DNA of pBR322 and core histones isolated from rat ascites hepatoma cells. Nucleosomal formation was estimated by analyzing the resulting DNA supercoils. When UVC-irradiated (3000 J/m²) DNA was used as a substrate for the nucleosome assembly system, the nucleosomal formation efficiency was reduced by half compared with nonirradiated DNA. On the other hand, when the reconstituted nucleosomes (minichromosomes) on the nonirradiated DNA were irradiated with UVC (3000 J/m²), about half each were disrupted and retained. These results indicate that it is difficult for UV-damaged DNA regions to supercoil around the histone octamers to form nucleosomes and that the histone octamers in the UV-damaged nucleosomes tend to be dissociated from DNA.     

Studies on responsiveness of hepatoma cells to catecholamines. V. Loss of adrenergic response of glycogen phosphorylase in rat ascites hepatoma AH130 cells

The beta-adrenoceptor-cyclic adenosine monophosphate (AMP) dependent glycogenolytic cascade was examined in normal rat hepatocytes and rat ascites hepatoma AH130 cells. The cyclic AMP content in AH130 cells was half of that in normal hepatocytes, and the cyclic AMP levels in both kinds of cells were clearly increased by isoproterenol (IPN). Cyclic AMP-dependent protein kinase activity was higher in AH130 cells than in normal hepatocytes. Phosphorylase kinase activities in 10000 x g supernatant of normal hepatocytes and AH130 cells were also increased in the presence of cyclic AMP. Phosphorylase a activities in the supernatant of both kinds of cells gradually decreased during incubation with 40 mM glucose at 37 degrees C, and the enzyme activity of normal hepatocytes was completely restored by the addition of Mg2(+)-adenosine triphosphate (ATP), but in the case of the hepatoma cells the recovery was small. The decreased phosphorylase a activity in the hepatoma cells was increased by additional glycogen but did not exceed the level before the incubation. In the case of normal hepatocytes it was not affected by glycogen. This indicates that glycogen contained in the cells influences the activation of phosphorylase; the glycogen content in AH130 cells was far less than in normal hepatocytes. On the other hand, when intact cells were incubated with a high concentration of glucose, phosphorylase a activity in the homogenate of normal hepatocytes was decreased and could be restored by IPN and dibutyryl cyclic AMP, but the enzyme activity in the homogenate of AH130 cells was very low and hardly changed after the incubation and treatment with these agents.(ABSTRACT TRUNCATED AT 250 WORDS)     

Optimization of the ion-pair high-performance liquid chromatographic separation of purine derivatives in erythrocytes, thymocytes and liver mitochondria

Various methods are described for the analysis of purine derivatives in biological samples by ion-pair high-performance liquid chromatography (HPLC) with both gradient and isocratic systems. A new approach is proposed that is suitable for the separation of nuclei acid constituents in different cells with a specific enzymatic activity pattern. The ion-pair HPLC methods were developed for the analysis of erythrocytes, lymphocytes and mitochondria acid-soluble fractions in clinical and experimental studies of normal and altered nucleotide metabolism. The results of studies of purine metabolite redistribution in mouse liver mitochondria during a 30-min incubation at 37 degrees C and data on purine metabolic alterations in mouse thymocytes during hepatoma growth are discussed.     

Cytotoxicity of metal 8-quinolinethiolates

High cytotoxicity has been established for the 8-quinolinethiolates of copper, cadmium, indium, antimony, bismuth, ruthenium, rhodium, palladium, osmium, iridium, and platinum on HT-1080 (human fibrosarcoma), MG-22A (mouse hepatoma), and B-16 (mouse melanoma) tumor cells. The greatest activity against HT-1080 was possessed by the iridium complex, and against MG-22A by the osmium complex. All the investigated metal 8-quinolinethiolates were highly toxic in relation to NIH 3T3 normal mouse embryo fibroblasts. __________ Translated from Khimiya Geterotsiklicheskikh Soedinenii, No. 6, pp. 870–873, June, 2006.     

CYTOTOXICITY AND MUTAGENICITY OF OPHTHALMIC SOLUTION PRESERVATIVES AND UVA RADIATION IN L5178Y CELLS

Four chemical preservatives commonly used in ophthalmic solutions were tested for their toxic and mutagenic potential in mouse lymphoma cells with and without exposure of the cells to ultraviolet A (UVA) radiation. The preservatives tested were benzalkonium chloride (BAK), chlorhexidine, thimerosal and ethylenediaminetetraacetic acid (EDTA). Cell survival and mutagenesis were measured using the L5178Y mouse lymphoma (TK +/-) system. Cells were exposed to varying amounts of preservatives for 1 h at 37 degrees C, and then aliquots were irradiated with UVA radiation (during the exposure to preservative). Cells were then assayed for survival, and for mutagenesis at the thymidine kinase (TK) locus. In concentrations commonly found in ophthalmic solutions, BAK, chlorhexidine, and thimerosal were toxic to cells, and thimerosal was slightly mutagenic. When cells were exposed to preservative and UVA radiation, chlorhexidine was mutagenic and the mutagenic activity of thimerosal was enhanced.     

Anti-migration effects of Gekko sulfated glycopeptide on human hepatoma SMMC-7721 cells

Gekko swinhonis Guenther has been used as an anti-cancer drug in traditional Chinese medicine for hundreds of years. Previous studies showed that the Gekko sulfated polysaccharide-protein complex suppressed the proliferation and migration of hepatoma cells. Gekko sulfated glycopeptide α was obtained from Gekko sulfated polysaccharide-protein complex using papain hydrolysis. Gekko sulfated glycopeptide α inhibited the proliferation and migration of SMMC-7721 cells. The secretion of IL-8 and the concentration of intracellular calcium were decreased after Gekko sulfated glycopeptide α exposure. SMMC-7721 cells in the control group showed abnormal features, with a polygonal shape, whereas this changed to a spindle shape after the treatment with Gekko sulfated glycopeptide α. Actin ?laments were distributed diffusely along the cell membrane in control cells, whereas those were polymerized and preferentially accumulated in the cytoplasm of treated cells. Microtubules distributed in the cytoplasm of untreated cells were located diffusely whereas those in treated cells were polymerized. Therefore, Gekko sulfated glycopeptide α inhibit the migration of hepatoma cells via reducing the secretion of IL-8 and the concentration of intracellular calcium, as well as regulating the reorganization of cytoskeleton.     

CRITICAL IMPORTANCE OF THE TRIPLET LIFETIME OF PHOTOSENSITIZER IN PHOTODYNAMIC THERAPY OF TUMOR

The relation between the lifetimes of the triplet states of various porphyrins and their photosensitizing effects on the photodynamic therapy (PDT) of tumor has been examined. Diethylene-triamine pentaacetic acid ester of 4-[1-(2-hydroxy-ethyloxy)ethyl]-2-vinyl deuteroporphyrin-IX gallium (III) complex (Ga-DP), zinc (II) complex (Zn-DP), and manganese (III) complex (Mn-DP) and Photofrin II (PII) are used as the photosensitizer. The triplet lifetimes have been measured for the samples adsorbed on filter paper (FP) and found to be 57 ms (Ga-DP), 26 ms (Zn-DP), less than or equal to 10 microseconds (Mn-DP) and 9 ms (PII). The phosphorescence of Ga-DP in tumor-bearing golden hamsters are measured both in tumor tissue and in liver. They show bi-exponential decay with the lifetimes of about 5 and 20 ms. From the values, the generation rate, kct[3O2], of singlet molecular oxygen in living animal tissue may be estimated to be an order of 10(2) s-1. The PDT effects have been quantitatively investigated for in vitro experiments; upon irradiation the growth inhibitions of mouse p388 leukemia cells are obtained as a function of concentration of Ga-DP, Zn-DP, Mn-DP and PII. The experimental results indicate that the PDT effects depend essentially on the triplet lifetimes of the photosensitizers.     

Dexmedetomidine promotes the progression of hepatocellular carcinoma through hepatic stellate cell activation

Dexmedetomidine (DEX) is an anesthetic that is widely used in the clinic, and it has been reported to exhibit paradoxical effects in the progression of multiple solid tumors. In this study, we sought to explore the mechanism by which DEX regulates hepatocellular carcinoma (HCC) progression underlying liver fibrosis. We determined the effects of DEX on tumor progression in an orthotopic HCC mouse model of fibrotic liver. A coculture system and a subcutaneous xenograft model involving coimplantation of mouse hepatoma cells (H22) and primary activated hepatic stellate cells (aHSCs) were used to study the effects of DEX on HCC progression. We found that in the preclinical mouse model of liver fibrosis, DEX treatment significantly shortened median survival time and promoted tumor growth, intrahepatic metastasis and pulmonary metastasis. The DEX receptor (ADRA2A) was mainly expressed in aHSCs but was barely detected in HCC cells. DEX dramatically reinforced HCC malignant behaviors in the presence of aHSCs in both the coculture system and the coimplantation mouse model, but DEX alone exerted no significant effects on the malignancy of HCC. Mechanistically, DEX induced IL-6 secretion from aHSCs and promoted HCC progression via STAT3 activation. Our findings provide evidence that the clinical application of DEX may cause undesirable side effects in HCC patients with liver fibrosis.Subject terms: Cancer microenvironment, Cell growth     

Cytotoxic di(8-quinolyl) disulfides

It has been shown that the nature of the substituent and its position in the quinoline ring markedly affects the antitumor activity and toxicity of di(8-quinolyl) disulfides. The greatest cytotoxicity in the series of methyl derivatives was shown by the 7-, 6-, and 3-isomers towards HT-1080 (human fibrosarcoma) and MG-22A (mouse hepatoma) tumor cells while the 2-methyl derivatives generally have no effect on these cells. High cytotoxicity was also shown (LC₅₀ <1 μg/ml) by other 7-substituted compounds (Cl, PhO, PhS) but they also appear to be highly toxic towards normal NIH 3Y3 mouse embryonic fibroblasts. A similar trend was observed in the series of 5-substituted compounds (NH₂, Cl, OMe, NO₂) which were highly active towards tumor cells but were toxic to normal cells. The best selectivity was found for the 6-substituted quinolines, the 6-methoxy derivative at low concentration brought about the death of tumor cells but appeared much less toxic towards normal fibroblasts (LC₅₀ 100 μg/ml with a corresponding LD₅₀ of 874 mg/kg ). __________ Translated from Khimiya Geterotsiklicheskikh Soedinenii, No. 5, pp. 750–754, May 2007.     

EFFECTIVENESS OF PHOTOFRIN II IN ACIWATION OF MACROPHAGES AND in vitro KILLING OF RETINOBLASTOMA CELLS

Abstract Administration of a small dose (300 ng/mouse) of photofrin II (PII) to mice, followed by 4 days of exposure to only ambient fluorescent light in animal quarters, induced Fc-receptor-mediated phagocytic and superoxide-generating capacities of peritoneal macrophages by five- and seven-fold, respectively. When these mice were kept in the dark for 4 days, no activation of macrophages was observed. These results suggest that macrophage activation is a consequence of photodynamic activation. Much higher doses (> 3000 ng/mouse) suppressed macrophage activity. However, 2 months after administration of 3000 ng PII/mouse, greatly enhanced phagocytic and superoxide-generating capacities of peritoneal macrophages were observed.
In vitro photodynamic activation of macrophages was analyzed after white or red fluorescent light exposure of mouse peritoneal cells (mixture of macrophages and B and T lymphocytes) in media containing PII. A short (10 s) white fluorescent light treatment of peritoneal cells in a medium containing 0.03 ng PII/mL produced the maximal level of phagocytic activity of macrophages. Illumination with the same total fluence of red fluorescent light requires a threefold higher concentration of PII to achieve the same extent of enhanced phagocytic activity of macrophages. Thus, photodynamic activation of macrophages with PII by white fluorescent light was more efficient than by red fluorescent light. Similarly, photodynamic killing of retinoblastoma cells was more efficient with white than red fluorescent light. The concentration of hematoporphyrin (HP) or PII required for direct photodynamic killing of retinoblastoma cells was roughly four orders of magnitude greater than that required for activation of macrophages. These results suggest that effective photodynamic therapy may be achieved with milder treatments that stimulate macrophage activity, an important component of immunopotentiation.     

Radiation-Induced Metabolic Shifts in the Hepatic Parenchyma: Findings from 18F-FDG PET Imaging and Tissue NMR Metabolomics in a Mouse Model for Hepatocellular Carcinoma

Purpose: By taking advantage of 18F-FDG PET imaging and tissue nuclear magnetic resonance (NMR) metabolomics, we examined the dynamic metabolic alterations induced by liver irradiation in a mouse model for hepatocellular carcinoma (HCC). Methods: After orthotopic implantation with the mouse liver cancer BNL cells in the right hepatic lobe, animals were divided into two experimental groups. The first received irradiation (RT) at 15 Gy, while the second (no-RT) did not. Intergroup comparisons over time were performed, in terms of 18F-FDG PET findings, NMR metabolomics results, and the expression of genes involved in inflammation and glucose metabolism. Results: As of day one post-irradiation, mice in the RT group showed an increased 18F-FDG uptake in the right liver parenchyma compared with the no-RT group. However, the difference reached statistical significance only on the third post-irradiation day. NMR metabolomics revealed that glucose concentrations peaked on day one post-irradiation both, in the right and left lobes—the latter reflecting a bystander effect. Increased pyruvate and glutamate levels were also evident in the right liver on the third post-irradiation day. The expression levels of the glucose-6-phosphatase (G6PC) and fructose-1, 6-bisphosphatase 1 (FBP1) genes were down-regulated on the first and third post-irradiation days, respectively. Therefore, liver irradiation was associated with a metabolic shift from an impaired gluconeogenesis to an enhanced glycolysis from the first to the third post-irradiation day. Conclusion: Radiation-induced metabolic alterations in the liver parenchyma occur as early as the first post-irradiation day and show dynamic changes over time.     

肝癌与肝硬化腹水微量元素Cu、Zn测定的临床诊断价值

腹水是一种病理性腹腔渗出液或漏出波．肝癌、肝硬化晚期，腹膜炎等多种疾病可致病人出现腹水，由于引起腹水病因不同，腹水中各物质含量亦有所不同．关于腹水中微量元素含量的研究尚少见报道。为此作者分三组测定了４８份腹水中的Ｃｕ、Ｚｎ含量，进行了分析比较，结果如下：１．Ｃｕ含量．其它组高于肝癌组，肝癌组高于肝硬化组，各组间差异非常显著，Ｐ＜０．０１。２．Ｚｎ含量，肝癌组高于肝硬化组，肝硬化组高于其它组，各组间差异显著，Ｐ＜０．０５．３．以上结果说明，测定腹水中Ｃｕ、Ｚｎ含量是一项了解腹水性质较敏感指标，尤其是肝硬化癌变时，Ｃｕ、Ｚｎ含量有明显的变化．     

DISTRIBUTION AND ELIMINATION OF PHOTOFRIN II IN MICE

The distribution and elimination of [14C]PII, the radioisotopically-labeled equivalent of the mixture of porphyrins known as Photofrin II used in the photodynamic treatment of solid tumors, were determined in tumor-free and SMT-F tumor-bearing DBA/2 Ha-DD mice. Following i.p. injection, drug was absorbed from the peritoneum with a half-life of about 1 h; elimination from plasma was rapid, declining about 1.4 logs in concentration over 48 h following i.v. administration. However, some [14C]-activity was still detectable after 75 days. Normal tissues take up the drug within about 7.5 h after administration, with peak concentrations distributed as follows: liver, adrenal gland, urinary bladder greater than pancreas, kidney, spleen greater than stomach, bone, lung, heart greater than muscle much greater than brain. Only skeletal muscle, brain, and skin located contralaterally to subcutaneously implanted SMT-F tumors had peak [14C]-activities lower than tumor tissue; skin overlying SMT-F tumors showed concentrations not significantly different (P greater than 0.3) from tumor. After 75 days all tissues examined retained some fraction of [14C]-activity, ranging from 16% for kidney to 61% for spleen, of the initial peak tissue levels. The primary route of elimination of Photofrin II was through the bile-gut pathway, with greater than 59% of the administered [14C]-activity recovered in the feces, and only about 6% in the urine, over 192 h. HPLC analyses of fecal extracts showed that mostly monomeric and other low molecular weight porphyrin components of Photofrin II were eliminated. The higher molecular weight oligomeric fractions of Photofrin II were retained in liver and spleen up to 14 days after injection.     

Cu(II)、Ni(II)配合物的合成、抗肿瘤作用及其对细胞周期分布的影响

以2-氨基-5-氯苯酚和2-喹啉甲醛合成的席夫碱作为配体,分别与氯化镍、氯化铜反应合成了2个金属配合物C1和C2,其结构通过单晶X-射线衍射进行了解析。采用MTT法测试了配合物C1和C2对不同人肝癌细胞系和正常肝细胞系HL-7702增殖抑制活性,结果表明C1、C2对人肝癌细胞系的抑制活性均优于顺铂,且对正常肝细胞系HL-7702的毒性要弱于顺铂。通过活性氧实验、细胞周期等实验,可以推断出配合物C1、C2抗肿瘤机制是通过产生活性氧造成肿瘤细胞的氧化损伤,以及使细胞周期停滞在G0/G1期阻滞细胞复制。     

THE EFFECTS OF ULTRAVIOLET AND VISIBLE RADIATION ON MOUSE ASCITES TUMOUR CELLS

Abstract— Effects of ultraviolet and visible radiation on the viability of Landschutz ascites tumour cells have been tested by growing control and treated tumour samples in adult mice. The tumour cells were irradiated as a dilute suspension in isotonic buffered salt solution, and were equilibrated at 0°C with oxygen or with nitrogen before irradiation.
Tumour cell proliferation was measured by a variety of techniques. The preferred assay-method was the growth of solid tumours in the axillae and groins of mice after sub-cutaneous inoculation of varying dilutions of treated or control ascites tumour cells. The immune response of the mice to the injected cells was reduced by whole body irradiation with a 300r dose of x-rays two days before inoculation. Results were calculated from parallel line assays using the reciprocal of the delay in appearance of the solid tumours up to 30 days post-innoculation. This reciprocal (1/T) was linearly related to the logarithm of the number of cells inoculated.
Photoreactivation has been demonstrated for this system, in which both U.V. and visible radiations were absorbed by the same cells. Light delivered alone in oxygen or in nitrogen was without effect on cell-viability, but it increased cell-survival after u.v.-irradiation in nitrogen and decreased survival after u.v.-irradiation in oxygen. Ultraviolet radiation alone was not significantly more lethal in oxygen than in nitrogen. A further observation in this work was an interaction between irradiated and control tumour cells injected into the same animal.
It is suggested that the radiation used may affect the antigenic character of the tumour cells as well as their reproductive capadity.     

水溶液中稀土离子与甘氨酰丙氨酸作用的NMR研究

利用¹H和¹³C NMR技术研究了水溶液中稀土离子与二肽甘氨酰丙氨酸(以下简称甘-丙二肽,记为GA)的配位作用。由稀土诱导位移的浓度依赖关系计算了Yb与甘-丙二肽配合物的稳定常数。测定了重稀土离子Dy³⁺、Ho³⁺、Er³⁺、Tr³⁺和Yb³⁺作用下GA的¹³C诱导位移,并根据Reuben方法对稀土诱导位移进行了线性相关分析。对配合物中配体骨架构象的模拟分析指出,Cl-C₂-N-C₃为旁式,C₂-N-C₃-C₄和C₅-C₂-N-C₃为反交叉式。系统比较了4种含甘氨酰二肽的侧基大小对配合物稳定常数、配体构象和配合物溶液结构的影响。