SMMC-7721肝癌细胞67kD层粘连蛋白受体的分离纯化

分离纯化肝癌细胞的 6 7kD层粘连蛋白受体 (6 7LR) ,以便进一步研究 6 7LR的结构、功能及其在肝癌浸润、转移过程中的作用 .以SMMC 772 1肝癌细胞和L 0 2正常肝细胞为材料 ,采用13 1I标记的层粘连蛋白测定其与细胞的结合能力 ;亲和层析法分离纯化层粘连蛋白受体 ,用SDS PAGE、放射自显影及体外竞争结合实验进行鉴定 .在相同条件下SMMC 772 1肝癌细胞与层粘连蛋白特异结合量为 17 5 4± 0 4 9ng 10 5细胞 ,而L 0 2正常肝细胞与层粘连蛋白的特异结合量为 8 36± 0 4 8ng 10 5细胞 .经过亲和层析 ,从SMMC 772 1肝癌细胞和L 0 2正常肝细胞均可获得纯化受体 ,SDS PAGE显示为单一条带 ,分子量为 6 7kD ,放射自显影及体外竞争结合实验表明其具有较强的与层粘连蛋白结合的活性 .体外竞争结合实验表明 ,SMMC 772 1肝癌细胞层粘连蛋白受体 (772 1LnR)的抑制率可达到 96 2 7± 2 2 9% ,而L 0 2正常肝细胞层粘连蛋白受体 (L 0 2LnR)的抑制率为 4 8 71± 3 79% ,这说明 772 1LnR与层粘连蛋白的亲和力明显高于L 0 2LnR(P <0 0 0 1) .结果表明 ,与L 0 2肝细胞比较 ,SMMC 772 1肝癌细胞具有与层粘连蛋白较强结合能力的特异受体 ,并从肝癌细胞膜上分离纯化到与层粘连蛋白有较强亲和力的 6 7LR     

Determination of the major zinc fractions in human serum by ultrafiltration

In this paper we report a method for measuring ultrafiltrable zinc in human serum by electrothermal atomic absorption spectrophtometry. We show also that ultrafiltration permits to determine alpha-2 macroglobulin bound zinc and losely bound zinc if a strong zinc ligand (EDTA) is added to serum before ultrafiltration. This last fraction, after deduction of ultrafiltrable zinc, represents roughly all albumin bound zinc. In 20 controls we found that ultrafiltrable zinc amounted 0.311 μmol/L (S.D.=0.117 μmol/L), alpha-2 macroglobulin bound zinc 3.08 μmol/L (S.D.=0.221 μmol/L), and albumin bound zinc 12.11 μmol/L (S.D.=1.95 μmol/L). Our method needs only a small volume of serum, it is simple and rapid but also very accurate and reliable. The losely bound fraction is very dynamic and, representing the physiologically active part of serum zinc, it could be a good marker of zinc deficiency.     

Effect of dietary nickel and iron on the trace element content of rat liver

The level and/or form of dietary iron, dietary nickel, and the interaction between them affected the trace element content of rat liver. Livers were from the offspring of dams fed diets containing 10–16 ng, or 20 μg, of nickel/g. Dietary iron was supplied as ferric chloride (30 μg/g) or ferric sulfate (30 μg, or 60 μg). In nickel-deprived rats fed 60 μg of iron/g of diet as ferric sulfate, at age 35 days, levels of iron and zinc were depressed in liver and the level of copper was elevated. At age 55 days, iron was still depressed, copper was still elevated, but zinc also was elevated. In rats fed 30 μg of iron/g of diet as ferric chloride, liver iron content was higher in nickel-deprived than in nickel-supplemented rats at 30, but not at 50, days of age. Also manganese and zinc were lower in nickel-deprived than in nickel-supplemented rats at age 35 days if their dams had been on experiment for an extended period of time (i.e., since age 21 days). Thus, the levels of copper, iron, manganese, and zinc in liver were affected by nickel deprivation, but the direction and extent of the affects depended upon the iron status of the rat.     

Two specific inhibitors of the phosphatidylinositol 3-kinase LY294002 and wortmannin up-regulate β1,4-galactosyltransferase I and thus sensitize SMMC-7721 human hepatocarcinoma cells to cycloheximide-induced apoptosis

Previous study indicated that β1,4-galactosyltransferase I (β1,4GT1) was up-regulated by cycloheximide (CHX) and thus enhanced apoptosis induced by CHX in SMMC-7721 cells. In this study, we reported that constitutively active Akt protein (myr-Akt) inhibited CHX-induced apoptosis in SMMC-7721 cells through down-regulating β1,4GT1. However, the two PI3K inhibitors LY294002 and wortmannin treatment up-regulated β1,4GT1 through enhancing Sp1 protein expression and consequently increased CHX-induced SMMC-7721 cells apoptosis. Besides, our results suggested that β1,4GT1 and cell surface galactose residues synthesized by elevated β1,4GT1 played an important role in SMMC-7721 cells apoptosis treated with CHX and PI3K inhibitor together. Moreover, we found that CHX accentuated β1,4GT1 through down-regulating Akt expression to mediate SMMC-7721 cells apoptosis. Taken together, PI3K inhibitors LY294002 and wortmannin up-regulated β1,4GT1 and enhanced CHX-induced apoptosis in SMMC-7721 cells, which suggested that PI3K inhibitors might have therapeutic potential when combined with CHX in the treatment of hepatoma.     

2′,4′-Dihydroxy-6′-methoxy-3′,5′-dimethylchalcone,from buds of Cleistocalyx operculatus,induces apoptosis in human hepatoma SMMC-7721 cells through a reactive oxygen species-dependent mechanism

Nowadays, much effort is being devoted to detect new substances that not only significantly induce the death of tumor cells, but also have little side effect on normal cells. Our previous study showed that 2′,4′-dihydroxy-6′-methoxy-3′,5′-dimethylchalcone (DMC) exhibited significant cytotoxic potential with an IC₅₀ value of 32.3 ± 1.13 μM against SMMC-7721 cells and could induce SMMC-7721 cells apoptosis. In the present study, we found that DMC was almost nontoxic to human normal liver L-02 and human normal fetal lung fibroblast HFL-1 cells as their IC₅₀ values (111.0 ± 4.57 and 152.0 ± 4.83 µM for L-02 and HFL-1 cells, respectively) were much higher. To further explore the apoptotic mechanism of DMC, we investigated the role of the reactive oxygen species (ROS) in the apoptosis induced by DMC in SMMC-7721 cells. Our results suggested that the cytotoxicity and the generation of intracellular ROS were inhibited by N-acetylcysteine (NAC). Reversal of apoptosis in NAC pretreated cells indicated the involvement of ROS in DMC-induced apoptosis. The loss of mitochondrial membrane potential (ΔΨm) induced by DMC was significantly blocked by NAC. NAC also prevented the decrease of Caspase-3 and -9 activities, the increase of Bcl-2 protein expression and the decrease of p53 and PUMA protein expressions. Together, these results indicated that ROS played a key role in the apoptosis induced by DMC in human hepatoma SMMC-7721 cells.     

Radiosensitization mechanism of riboflavin in vitro

Riboflavin, suggested to be a radiosensitizer, was studied in murine thymocytes and human hepatoma L02 cell line in vitro with MTT method and fluorescence microscopy. When the murine thymocytes treated with 5–400 μmol/L riboflavin were irradiated by 5 Gy ⁶⁰Co γ ionizing radiation, the low concentration groups, i.e. treated with 5–50 μmol/L riboflavin, showed a different surviving fractions-time relating correlation compared with the high concentration groups, i.e. treated with 100–400 μmol/L riboflavin. The former had a high survival level at the end of irradiation, but which, after 4-h incubation, decreased rapidly to a low level. On the contrary, the high concentration groups showed a low survival level at the end of irradiation, and a poor correlation was found between the surviving fraction and the incubation time, after 4 h a little difference was observed. The results of fluorescence microscopy indicated that under low concentration conditions, the riboflavin localized mainly in nucleus (both perinuclear area and inside of nuclear membrane), while under high concentration conditions, intensive riboflavin also localized around cytoplasmic membranes. Thus we can conclude: the riboflavin had radiosensitivity effect on DNA under low concentration conditions, and enhanced the damage to cytoplasmic membrane under high concentration conditions. Also the most effective concentration of riboflavin can be evaluated to be approximate 100 μmol/L.     

The changes of metabolism balance of zinc and copper in gastric juice with widely varying dietary zinc intake

The concentrations of zinc and copper in gastric juice of humans who had widely varying dietary zinc intake were evaluated. In order to compare this with zinc and copper levels of normal dietary individuals, we also determined the zinc and copper levels in healthy individuals' plasma and in cancer patient's natural tissue, all of whom had normal diets. The correlation coefficients between zinc and copper were 0.71, 0.45, and 0.55, respectively, in gastric juice, plasma, and tissue of normal dietary subjects. Such correlation changed and was destroyed when there was a high zinc level in gastric juice. When gastric juice zinc level changed from mean value 16.8 μmol/L to 262.5 μmol/L, the correlation coefficient varied from 0.71 to −0.04, and the copper level also varied from mean value 8.96 μmol/L to 4.89 μmol/L. These findings probably give the evidence to suggest that a high zinc level will restrain the copper level and break the balance of the human body's zinc and copper metabolism.     

Effects of a multivitamin mineral supplement on zinc and copper status during pregnancy

The effect of a multivitamin-mineral supplement was investigated during pregnancy according to a double-blind protocol by determining zinc and copper in maternal plasma, mononuclear and polynuclear zinc and copper at the third, sixth, eighth, and ninth months of gestation. The subjects were supplemented from the first trimester until delivery. A significant decrease was observed in plasma zinc that varied from 11.5 μmol/L to 10.8 μmol/L in the supplemented group (n=29) and from 11 μmol/L to 10 μmol/L in the placebo group (n=33) at 3 and 9 mo of gestation, respectively. In contrast, plasma copper levels increased in a way depending upon the stage of gestation in both groups: from 24.7 to 28.2 μmol/L in the treated group and from 24.9 to 30.9 μmol/L in the placebo group at 3 and 9 mo of gestation, respectively, but the difference was only significant in the placebo group. No difference between groups was observed in mononuclear and polynuclear zinc or copper levels. These trace elements were also determined in cord blood at delivery. There were no statistically significant differences in zinc and copper concentration found in placebo group and supplemented group. Finally, the beneficial effect of supplementation on muscular cramps and appearance of vergetures was noted.     

Some differentiating effects of selenium on the cultured human hepatoma cells and human pulmonary adenocarcinoma cells in vitro

Two human hepatoma cell lines, QGY-7703 and SMMC-7721, and two human pulmonary adenocarcinoma cell lines, LTEP-a-2 and SPC-A-1, were found to respond to 1 μg/mL Na₂SeO₃, 24 h, in-vitro treatment by decreasing its confluent saturation density. The same treatment was found to cause an increase in the adhesiveness of cells measured as resistance to detachment by trypsin/EDTA. The pathological features of tumors after heterotransplantation of treated and untreated cells were similar, but the size of tumor grown from treated cells was much smaller.     

Serum extracellular superoxide dismutase activity as an indicator of zinc status in humans

The present study focused on whether serum extracellular superoxide dimutase (EC-SOD) activity can be used as a functional indicator of marginal zinc deficiency in humans. Subjects in this study were 444 healthy adults over 30 yr of age living a normal rural life in Kyunggi province, Korea. The mean dietary zinc intake of subjects obtained from one 24-h recall was 6.41 ± 4.35 mg and the average serum zinc concentration of the subjects was 11.06 ± 2.44 (μmol/L. Subjects were divided into three groups by serum zinc concentrations: adequate (serum zinc >10.7 (μmol/L), low (serum zinc 9.0–10.7 μmol/L), and very low (serum zinc <9.0 μmol/L) groups. A total of 50 subjects were selected from the three groups for analysis of EC-SOD activities. The EC-SOD activity of subjects increased with increasing serum zinc concentrations, and the activities of the three groups were significantly different as indicated by the Kruskal-Wallis test (p = 0.0239). Also, serum EC-SOD activities were significantly correlated with serum zinc concentrations (r = 0.289,p = 0.04). Serum EC-SOD activities, however, were not significantly correlated to the dietary zinc intakes. In conclusion, these results show that EC-SOD activities are decreased in subjects with low serum zinc concentrations and suggest that EC-SOD activity may be a functional indicator of zinc nutritional status in humans.     

The inhibitory effect of transthyretin gene on growth of human hepatoma cells

Transthyretin(TTR) gene was highly expressed in normal liver and it has been found to be deleted in part of DNA samples from human hepatic cancer.Its mRNA expression was suppressed in most hepatoma samples.In order to study the biological effect of TTR gene on the growth of hepatoma cells,a recombinant vector containing TTR cDNA was constructed by pCMV,then it was transfected into hepatoma cell lines SMMC-7721 and Q3.It has been demonstrated that the inhibition of growth rate of TTR cDNA transfected hepatoma cells was about 50% in strength compared with that of the control.This inhibition was further enhanced when the transfected hepatoma cells were treated with all-trans retinoic acid.Hepatoma cells of cell lines PLC/PRF/5,SMMC-7721 and Q3 as well as hepatoma cells SMMC-7721 transfected with pCMV or pCMV-TTR were analyzed for TTR expression by Northern hybridization.The low level of TTR expression was found in both hepatoma cell lines and in SMMC-7721 cells transfected with pCMV alone.However,a remarkable TTR mRNA expression was observed in hepatoma SMMV-7721 cells transfected with pCMV-TTR.It seems possible that TTR gene might be a candidate of cancer suppressor gene for human hepatic cancer.     

SeO2 induces apoptosis with down-regulation of Bcl-2 and up-regulation of P53 expression in both immortal human hepatic cell line and hepatoma cell line

An immortal human hepatic cell line HL-7702 and human hepatoma cell line SMMC-7721 were treated with 3–30 μM SeO₂. SeO₂ at 30 μM markedly inhibited cell proliferation and viability, and prompted apoptosis of both normal hepatic and hepatoma cells after 48 h treatment. SeO₂ could also down-regulate the Bcl-2 level, greatly in HL-7702 and slightly in SMMC-7721 cells, but up-regulate wild type P53 level a little in HL-7702 and significantly in SMMC-7721 cells. The Bcl-2/P53 value was closely correlated with the apoptotic rate as well as SeO₂ concentrations.     

Effects of diabetes type and treatment on zinc status in diabetes mellitus

Zinc status was assessed in 53 diabetic patients: 18 insulin-dependent diabetic patients (IDDM), 22 noninsulin-dependent diabetic patients (NIDDM) treated with oral antidiabetic agents, and 13 insulin-treated, noninsulin-dependent diabetic patients (IRDM). Plasma zinc concentrations were in the usual range for healthy subjects in these three groups (15.3±0.9 μmol/L). Urinary zinc excretions were elevated in the IDDM group (18.3±4.1 μmol/24 h;p<0.01 vs normal) and in the NIDDM group (17.5±3.5 μmol/24 h;p<0.01 vs normal), but normal in the IRDM group (11.3±2.4 μmol/24 h). In 14 NIDDM patients treated with transient continuous sc insulin injections, urinary zinc decreased from 16.5±2.2 μmol/24 h before insulin treatment to 11.5±0.3 μmol/24 h after insulin treatment without any modification in plasma zinc concentrations.     

Expression of tachykinin receptors in Xenopus oocytes injected with poly(A)+RNA from cat dorsal root ganglion

The expression of the types of tachykinin receptors in the dorsal root ganglion (DRG) neurons by means ofXenopus oocyte expressing system was studied. Poly(A)⁺ RNAs were extracted from cat cervical and lumbar DRG. Two days after injection of Poly (A)⁺ RNAs, the oocytes were recorded with the two-electrode voltage clamp technique. In the oocytes injected with DRG poly(A)⁺ RNA, [Sar⁹, Met(O₂)¹¹]-substance P(Sar -SP, 1 μmol/L), neurokinin A (NKA, 1 μmol/L) or [β-Ala⁸]-neurokinin A₍₄₋₁₀₎ (Ala-NKA, 1 μmol/L) produced an inward current comprising a rapid spike and a long sustained oscillatory component for several minutes. Sar-SP induced response was blocked by NK-1 antagonist L-668, 169 (1 μmol/L), but not by NK-2 antagonist L-659, 877(1μmol/L). In contrast, Ala-NKA and NKA responses were only blocked by L-659, 877. The oocytes injected with DH Poly(A)⁺RNA also responded to Sar-SP and NKA with similar inward currents, which were selectively blocked by L-668, 169 and L-659, 877, respectively. These tachykinins-induced responses had a potent desensitization. The present data indicate expression of NK-1 and NK-2 receptors in DRG neurons, suggesting that there may be tachykinin autoreceptors on the nociceptive primary afferent terminals. Project supported by the National Natural Science Foundation of China (Grant No. 39370249).     

Cadmium induces mitochondria-dependent apoptosis of normal human hepatocytes

The heavy metal cadmium, an environmental pollutant, has been widely demonstrated to be toxic, in particular for liver. In murines, cadmium induces apoptosis of hepatocytes and hepatomas. In human cells, apoptosis induced by cadmium has been exclusively demonstrated in tumoral cell lines. Nothing was known in normal liver, in vitro or in vivo. In the present study, we examined the effects of cadmium in nonmalignant human hepatocytes. For that purpose, we investigated whether cadmium was able to induce apoptosis of normal human hepatocytes (NHH) in primary culture and of a SV40-immortalized human hepatocyte (IHH) cell line. Treatment of IHH and NHH with cadmium induced the presence of a sub-G₁ population at 10 and 100 μmol/L, respectively. DAPI staining of both cell types treated with cadmium 100 μmol/L revealed the induction of nuclear apoptotic bodies, supporting the hypothesis of apoptosis. In IHH and NHH, cadmium 100 μmol/L induced PARP cleavage into a 85 kDa fragment. In order to investigate the involvement of mitochondria in cadmium-induced apoptosis, we measured the mitochondrial membrane potential (Δ_Ψm). We observed that in IHH and NHH, cadmium 100 μmol/L induced a decrease of Δ_Ψm. As expected, cadmium under the same conditions enhanced caspase-9 and caspase-3 activities. In addition, cadmium from 1 to 100 μmol/L induced the expression of p53 and phosphorylation of its Ser15 in IHH and NHH. In conclusion, we showed in this study that human hepatocytes were sensitive to cadmium and apoptosis induced at concentrations suggested in the literature to inhibit p53 DNA-binding and DNA repair.     

自养小球藻多糖对人肝癌细胞SMMC-7721凋亡的诱导

应用超声破碎碱提法制备自养小球藻多糖。在体外培养条件下,用不同浓度的小球藻多糖处理肝癌细胞,并通过MTT,双苯并咪唑(Hoechst33258)染色,免疫细胞化学等方法检测。结果表明,1g/L的小球藻多糖作用肝癌细胞SMMC-77211 2h,对其增殖有显著抑制作用,且能通过下调抗凋亡蛋白Bcl-2及上调凋亡执行蛋白Capase-3的表达来诱导其发生凋亡。     

Human augmenter of liver regeneration is important for hepatoma cell viability and resistance to radiation-induced oxidative stress

To gain new insight into the biological function of the human augmenter of liver regeneration (hALR) in HCC, we studied its involvement in radiation-induced damage and recovery of HCC cells. We found that hALR expression was up-regulated in both HCC tissues and multiple hepatoma cell lines and correlated significantly with increased radiation clonogenic survival after radiation treatment. Exogenous expression of hALR increased radiation resistance in SMMC-7721 cells, and the increased survival was accompanied by a decrease in apoptosis and a prolonged G₂–M arrest after irradiation. Overexpression of ALR significantly increased the mitochondrial membrane potential, inhibited cytochrome c release, and opposed the loss of intracellular ATP levels after radiation. Moreover, knockdown of ALR by siRNA resulted in inhibition of viability in the absence of exogenously added oxidative stress and radiation sensitization in HepG2 cells. Importantly, hALR expression was very low in normal hepatocyte L02 cells, and hALR silencing had a minimal effect on L02 viability and radiation sensitivity. These results suggest that human ALR is important for hepatoma cell viability and involved in the protection of hepatoma cells against irradiation-induced damage by its association with mitochondria. Targeting hALR may be a promising novel approach to enhance the radiosensitivity of hepatoma cancers.     

Silibinin pretreatment protects against Ochratoxin A-mediated apoptosis in primary rat hepatocytes

The inhibitory effect of silibinin on ochratoxin A (OTA)-mediated apoptosis on primary rat hepatocytes was investigated. Rat hepatocytes were prepared by two different methods: the classical enzymatic digestion method by collagenase perfusion and a new EDTA-perfusion method. The EDTA-perfusion method yielded hepatocytes, which were stably cultivated without DNA fragmentation for up to 96 h, whereas the collagenase-prepared hepatocytes showed apoptosis events as early as from the start of preparation even in the absence of OTA. Treatment with 12.5 μmol/l OTA of cultured hepatocytes prepared under ETDA perfusion developed DNA-laddering after 24–36 h. Lipopolysaccharide (LPS) of 0.1 up to 12.5 μg/ml showed no apoptotic DNA-effects under these conditions. A low concentration of 26 μmol/l silibinin given prior to OTA slightly prevented OTA-mediated DNA-laddering, whereas a five times higher concentration of silibinin (130 μmol/l) completely inhibited OTA-mediated apoptosis. Under the same conditions, caspase-3 activity in hepatocytes increased in a time-dependent manner under OTA exposure within 12–24 h but was blocked by 130 μmol/l silibinin. In contrast, LPS incubation for 12 and 24 h did not alter caspase-3 activity. To measure viability of OTA-/LPS-treated hepatocytes, the MTT-test and Live/Dead kit were applied. The results demonstrated that the used OTA concentration of 12.5 μmol/l only moderately decreased viability for up to 24 h but showed cytotoxic effects depending on longer incubation times (≥36 h). In contrast, LPS up to 12.5 μg/ml exhibited no cytotoxic effects up to 48 h. In summary, our results showed contrasting effects on apoptosis in primary rat hepatocytes by OTA (produces apoptosis) versus LPS (produces no apoptosis), also depending on the method of hepatocyte preparation. Silibinin at 130 μmol/l showed significant hepatoprotective and antiapoptotic effects against OTA-mediated cell damage on cultured rat hepatocytes.     

The reproducibility of the plasma response to a physiological dose of zinc in healthy subjects

Zinc absorption may be estimated by measuring the area under the plasma zinc curve following the ingestion of a zinc supplement. The aim of this study is to determine the reproducibility of such a response when a small dose of zinc is administered to healthy volunteers. Five female subjects were asked to consume 4.5 mg elemental zinc, and blood samples were obtained at 30 min intervals for 5 h. The experiment was repeated in the same volunteers 12–16 d later. The area under the plasma zinc curve was 30% lower after the second zinc tolerance test compared with the first (11.0 vs 15.8 μmol/1 h). This difference could not be explained by differences in the fasting plasma zinc levels (12.9 μmol/L Experiment one, 15.1 μmol/L Experiment 2) nor was it related to technical or clinical parameters. The area under the curve after 5 h was strongly correlated with the response after 4 h. Hence we conclude that a small dose of zinc can be used to determine zinc absorption and a shorter experimental period may be used. However, trials must be designed to take into account the sequence variability in the response.