Polyamine metabolism in primary human colon adenocarcinoma cells (SW480) and their lymph node metastatic derivatives (SW620)

The natural polyamines are multifunctional constituents of all eucaryotic cells. The objective of this work was to compare aspects of polyamine metabolism in two related cell lines with the idea to investigate whether metabolic differences can be attributed to functional differences of the cells. The human colon carcinoma-derived cell lines SW480 and SW620 were chosen as models. SW480 cells were isolated from the primary tumour, SW620 cells from a lymph node of the same patient. SW620 cells grow faster, and the key regulatory enzymes of polyamine biosynthesis (ODC and AdoMetDC) are more active in the metastatic cells. Moreover, their ability to accumulate polyamines from the environment is more important than of SW480 cells. Likewise polyamine concentrations were markedly higher in SW620 cells, although they are much smaller than SW480 cells, and have a particularly small cytoplasmic space. Both cell lines show a striking diminution of ODC and AdoMetDC activities and changes in the polyamine patterns at the transition from exponential to non-exponential growth--most probably as a consequence of high cell density. Depletion of putrescine and spermidine due to inactivation of ODC by DFMO causes accumulation of cells in G1, and a proportional decrease of S-phase cells in both cell lines. Based on morphologic and other criteria SW480 and SW620 cells were typified as poorly differentiated. In agreement with their low grade of differentiation they exhibit a low alkaline phosphatase activity. However, the time-dependent decrease of alkaline phosphatase is not typical of differentiation patterns of other adenocarcinoma-derived cell lines or of normal enterocytes. The high capacity of de novo polyamine biosynthesis and of polyamine uptake is presumably a prerequisite for the rapid growth and invasiveness. The fact that these properties were more accentuated in the case of SW620 cells and paralleled enhanced metastatic properties indicate relationships between basic parameters of polyamine metabolism and malignancy.     

Spectral karyotyping of the human colon cancer cell lines SW480 and SW620

The cell lines SW480 and SW620, derived from different stages of colon carcinoma in the same patient, have been used for a number of biochemical, immunological, and genetic studies on colon cancer. A comparative analysis of their karyotypes may identify chromosomal aberrations that might represent markers for metastatic spread. In the present study spectral karyotyping (SKY) was applied to these two colon cancer cell lines. Compared to previously reported G-banded karyotypes, 9 (SW480) and 7 (SW620) markers were identical, 3 (SW480) and 3 (SW620) markers could be redefined, 5 (SW480) and 8 (SW620) markers were newly identified, and 4 (SW480) and 5 (SW620) of the previous described markers could not be confirmed. The redefined aberrations include very complex rearrangements, such as a der(16) t(3;16;1;16;8;16; 1;16;10) and a der(18)t(18;15;17)(q12; p11p13;??) in SW620 and a der(19)t(19;8;19;5) in SW480, that have not been identified by conventional banding techniques. The resulting chromosome gains (5q11-->5q15, 7pter-->q22, 11, 13q14-->qter, 20pter-->p12, X) and losses (8pter-->p2, 18q12-->qter, Y) found in both SW480 and SW620 were in good agreement with those frequently described in colorectal tumors as primary changes in the stem cell. Abnormalities found exclusively in SW620 cells only (gains of 5pter-->5q11, 12q12-->q23, 15p13-->p11, and 16q21-->q24 and losses of 2pter-->2p24, 4q28-->qter, and 6q25-->qter) can be viewed as changes that occurred in a putative metastatic founder cell.     

Cytotoxic effects of the polyamine oxidase inactivator MDL 72527 to two human colon carcinoma cell lines SW480 and SW620

N ¹,N ⁴-bis(2,3-butadienyl)-1,4-butanediamine (MDL 72527) was considered to be a selective inactivator of FAD-dependent tissue polyamine oxidase. Recently MDL 72527 was reported to induce apoptosis in transformed hematopoietic cells through lysosomotropic effects. Since it is the only useful inhibitor of polyamine oxidase available at present, the re-evaluation of its properties seemed important. Human colon carcinoma-derived SW480 cells and their lymph node metastatic derivatives (SW620) were chosen for our study because they differ in various aspects of polyamine metabolism but have similar polyamine oxidase activities. MDL 72527 inhibited cell growth in a concentration-dependent manner, depleted intracellular polyamine pools, and caused the accumulation of N ¹-acetyl derivatives of spermidine and spermine. SW620 cells were more sensitive to the drug than were SW480 cells. At 150 μmol/L MDL 72527, SW620 cells accumulated in S-phase of the cell cycle, showed decreased polyamine transport rate, and showed no increase of polyamine N ¹-acetyltransferase activity. In contrast, SW480 cells were not arrested in a particular phase of the cell cycle, showed enhanced polyamine uptake, and showed a mild induction of acetyltransferase. The results suggest that MDL 72527 retains its value as a selective tool in short-term experiments only at concentrations not exceeding those necessary for the inactivation of polyamine oxidase. At concentrations above 50 μmol/L and at exposure times longer than 24 h, it may derange cell functions nonspecifically, and thus blur the results of studies intended to elucidate polyamine oxidase functions. This revised version was published online in July 2006 with corrections to the Cover Date.     

Serum protease inhibitors promote anchorage-independent growth of transformed cells in serum-free medium.

The effect of three serum serine protease inhibitors on the serum-free agar growth of an SV40-transformed 3T3 cell line was investigated. Antithrombin III, alpha-2-macroglobulin and alpha-1-antitrypsin were found to potently stimulate colony growth in a semisolid medium because of their anti-proteolytic properties. These results indicate that protease inhibitors can facilitate tumor cell growth in serum-free agar cultures and suggest that the stimulatory effect of serum on the growth of certain transformed cells in agar may at least partially be due to the high levels of protease inhibitors found in serum.     

Epidermal growth factor effect on serum-free growth of primary and metastatic human tumors

The effect of epidermal growth factor (EGF) on the in vitro growth of human malignant tumors was compared in serum-supplemented (n = 54) and serum-free (N = 41) media at clonal density to determine the true EGF dependency of tumors. In the complete absence of serum at a 1,000 cells/cm2 seeding inoculation (approximately 100-200 adherent cells), EGF increased growth by greater than 50% in 27 of 41 specimens (66%), and growth increased by 100% or more in 18 of these EGF-sensitive tumors. In 12 serum-free cultures (29%), in vitro growth failed to occur without EGF. With 10% serum supplementation and a lower cell density (250 cells/cm2), EGF increased growth by greater than 50% in 34 of 54 specimens (63%), of which 25 had more than a 100% increase. The maximum growth induced by EGF in serum was usually seen in those tumors already capable of moderate in vitro growth. No difference in response to EGF was detected between specimens from primary tumors (n = 24) and those from metastases (n = 30). Under the stringent culture conditions of complete absence of serum and with tumors seeded at a low cell number, EGF stimulated most primary or metastatic human tumors to establish and sustain short-term in vitro growth successfully.     

Resveratrol induces mitochondrial respiration and apoptosis in SW620 colon cancer cells

BackgroundThe polyphenol resveratrol (RSV) is found in the skin of red grapes and has been reported to exhibit anticancer properties. The antitumor effects of RSV in the gastrointestinal tract have gained considerable interest due to the high exposure of this tissue to this dietary compound. One of the hallmarks of cancer cells is their particular metabolism mainly relying on glycolysis for ATP production rather than mitochondrial oxidative phosphorylation. Although RSV has been described to act as a calorie-restriction mimetic, modulating energy metabolism in normal tissues, little efforts have been done to study the effects of this polyphenol in the metabolism of cancer cells. Taking this into account, the aim of this study was to explore metabolic effects of this polyphenol in colon cancer.MethodsOxygen consumption, ATP levels, Western blotting and other molecular biology techniques were carried out to characterize the metabolic signature of RSV in SW620 colon cancer cells.ResultsParadoxically, the cytotoxic effects of RSV were associated with an increase in oxygen consumption supported by mitochondrial biogenesis and increased fatty acid oxidation. This partial reversion of the Warburg effect was followed by hyperpolarization of mitochondrial membrane and ROS production, leading to an increased apoptosis.ConclusionsOur results propose that the anticancer mechanisms of RSV could reside in targeting cancer cell metabolism, promoting mitochondrial electron transport chain overload and, ultimately, increasing ROS production.General significanceThese results shed new light into the anticancer mechanism of RSV supporting the ability of this compound in potentiating the effects of chemotherapy.     

Selective growth of normal adult human urothelial cells in serum-free medium

Summary A serum-free medium (HMRI-2) has been developed for the outgrowth and subculture of epithelial cells from normal adult human ureter and bladder. Medium HMRI-2 consists of Ham’s MCDB 152 with double the amounts of the essential amino acids in Stock 1, low Ca²⁺ (0.06 mM) and is supplemented with epithelial growth factor, 5 ng/ml; transferrin, 5 μg/ml; insulin, 5 μg/ml; ethanolamine and phosphoethanolamine, 0.1 mM each; hydrocortisone, 2.8×10⁻⁶ M; and bovine pituitary extract, 126 μg protein/ml. The cultured cells showed ultrastructural markers of epithelial cells (prekeratin fibers, tonofilaments, surface microvilli with glycocalyx), exhibited ABO antigens, and had a normal human diploid karyotype. Primary cultures could be subcultured and also cryopreserved in HMRI-2 in liquid nitrogen. Cells in mass cultures showed a population doubling time of 40.5±4.5 h and had a maximum in vitro life span of 20 to 25 population doublings. It was observed that primary outgrowths, secondary cultures, and even cryopreserved cells all retained the capacity to respond to high Ca²⁺ and serum by differentiation and desquamation. This study has resulted in the availability of easily obtainable serum-free epithelial cultures from normal adult human ureter and bladder. The useful in vitro life span of these cultures may be important in future studies of carcinogenesis. This work was supported by a grant from the National Cancer Institute (R01CA25089), Bethesda, MD.     

Hyperosmotic stress up-regulates the expression of major vault protein in SW620 human colon cancer cells

The major vault protein (MVP) is the major constituent of the vault particle, the largest ribonuclear protein complex described to date and is identical to lung resistance-related protein (LRP). Although MVP is also expressed in several normal tissues, little is known about its physiological role. MVP played a protective role against some xenobiotics and other stresses. We thus investigated the effect of osmotic stress on MVP expression by treating human colon cancer SW620 cells with sucrose or NaCl. The expression level of both MVP protein and MVP mRNA was increased by the osmostress. Sucrose or sodium chloride could also enhance MVP promoter activity. Inhibition of p38 MAPK in SW620 cells by SB203580 inhibited the expression of MVP under hyperosmotic stress. These findings suggested that osmotic stress up-regulated the MVP expression through p38 MAPK pathway. Down-regulation of MVP expression by MVP interfering RNA (RNAi) in SW620 cells increased the sensitivity of the cells to hyperosmotic stress and enhanced apoptosis. Furthermore, MVP RNAi prevented the osmotic stress-induced, time-dependent increase in phosphorylated Akt. These findings suggest that the PI3K/Akt pathway might be implicated in the cytoprotective effect of MVP.Our data demonstrate that exposure of cells to hyperosmotic stress induces MVP that might play an important role in the protection of the cells from the adverse effects of osmotic stress.     

Novel irreversible EGFR tyrosine kinase inhibitor 324674 sensitizes human colon carcinoma HT29 and SW480 cells to apoptosis by blocking the EGFR pathway

Background

Epidermal growth factor receptor (EGFR) is widely expressed in multiple solid tumors including colorectal cancer by promoting cancer cell growth and proliferation. Therefore, the inhibition of EGFR activity may establish a clinical strategy of cancer therapy.

Methods

In this study, using human colon adenocarcinoma HT29 and SW480 cells as research models, we compared the efficacy of four EGFR inhibitors in of EGFR-mediated pathways, including the novel irreversible inhibitor 324674, conventional reversible inhibitor AG1478, dual EGFR/HER2 inhibitor GW583340 and the pan-EGFR/ErbB2/ErbB4 inhibitor. Cell proliferation was assessed by MTT analysis, and apoptosis was evaluated by the Annexin-V binding assay. EGFR and its downstream signaling effectors were examined by western blotting analysis.

Results

Among the four inhibitors, the irreversible EGFR inhibitor 324674 was more potent at inhibiting HT29 and SW480 cell proliferation and was able to efficiently induce apoptosis at lower concentrations. Western blotting analysis revealed that AG1478, GW583340 and pan-EGFR/ErbB2/ErbB4 inhibitors failed to suppress EGFR activation as well as the downstream mitogen-activated protein kinase (MAPK) and PI3K/AKT/mTOR (AKT) pathways. In contrast, 324674 inhibited EGFR activation and the downstream AKT signaling pathway in a dose-dependent manner.

Conclusion

Our studies indicated that the novel irreversible EGFR inhibitor 324674 may have a therapeutic application in colon cancer therapy.     

Plectin regulates invasiveness of SW480 colon carcinoma cells and is targeted to podosome-like adhesions in an isoform-specific manner

Co-ordination of cytoskeletal networks and their dynamics is an essential feature of cell migration and cancer cell invasion. Plectin is a large cytolinker protein that influences tissue integrity, organisation of actin and intermediate filaments, and cell migration. Alternatively spliced plectin isoforms are targeted to different subcellular locations. Here, we show that plectin ablation by siRNA impaired migration, invasion and adhesion of SW480 colon carcinoma cells. A previously less well characterised plectin isoform, plectin-1k, co-localised with epithelial integrins, N-WASP, cortactin, and dynamin in podosome-like adhesions in invasive SW480 colon carcinoma cells. Transfection of alternative plectin N-terminal constructs demonstrated that the first exons of isoforms 1k, 1 and 1d can target the actin-binding domain of plectin to podosome-like adhesions. Finally, Plectin-1k N-terminus rescued adhesion site formation in plectin knock-down cells. Thus, plectin participates in actin assembly and invasiveness in carcinoma cells in an isoform-specific manner.     

Both calcium and ROS as common signals mediate Na(2)SeO(3)-induced apoptosis in SW480 human colonic carcinoma cells

Recent studies have shown that reactive oxygen species (ROS) play a crucial role in Se-induced cell apoptosis. A number of studies have demonstrated that perturbed cellular calcium homeostasis has been implicated in apoptosis. The main objective of this study was to evaluate the role of Ca(2+) in Na(2)SeO(3)-induced apoptosis and the relationship between Ca(2+) and ROS in human colonic carcinoma cells SW480. When SW480 cells were exposed to 25-100 microM Na(2)SeO(3), both cell apoptosis and growth inhibition were observed by flow cytometric analysis and 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay. Na(2)SeO(3) was able to induce increase of [Ca(2+)](i) and ROS production and disrupt mitochondrial membrane potential (Delta Psi m) in SW480 cells monitored by using a confocal laser scanning microscope. Ca(2+) channel inhibitor CoCl(2) and an intracellular Ca(2+) chelator o-phtalaldehyde, 1,2-bis(2-aminophenoxy)-ethane-N,N,N',N'-tetra-acetic acid acetoxymethyl ester (BAPTA) completely inhibited [Ca(2+)](i) increase, but catalase had no effect on Na(2)SeO(3)-induced increase of [Ca(2+)](i). BAPTA-AM, CoCl(2), and mitochondrial Ca(2+) uptake inhibitor ruthenium red blocked Delta Psi m dissipation. The increase of ROS was also suppressed by CoCl(2), BAPTA, ruthenium red, N-acetylcysteine and catalase, respectively. The mitochondrial uncoupler carbonyl cyanide p-(trifluoromethoxy) phenylhydrazone (FCCP) completely inhibited Na(2)SeO(3)-induced ROS increase. This showed that ROS increase is due to mitochondrial Ca(2+) overload. The Na(2)SeO(3)-induced apoptosis of SW480 cells was also inhibited by CoCl(2), BAPTA, ruthenium red, N-acetylcysteine, and catalase, respectively. The results mentioned above imply that both calcium and Ca(2+)-dependent ROS as a signal molecule mediate apoptosis induced by Na(2)SeO(3) in SW480 cells.     

Methods for growth of cultured cells in serum-free medium

Mitochondria prepared from skeletal muscle are typically contaminated with sarcoplasmic reticulum fragments and salt-soluble proteins. These contaminants can be removed by density-gradient centrifugation in Percoll. The resulting mitochondria retain both their original state three respiratory rates and their respiratory control ratios. The method has also been adapted to prepare mitochondria from very small tissue samples.     

Dextran T-500 improves survival and spreading of chick embryo cells in serum-free medium

The supplementation of serum-free Eagle's minimal essential medium (EMEM) with Dextran T-500 significantly improves attachment, spreading and survival of chick embryo cells in primary (myoblasts) and secondary (fibroblasts) cultures. These effects were observed in quiescent cultures incubated at 4 degrees C as well as at 37 degrees C. The results suggest that dextran can be used to simplify conditions for research on factors influencing cell proliferation and differentiation in serum-free media.     

Modulation of c-myc by transforming growth factor-beta in human colon carcinoma cells

Previous work indicated that transforming growth factor-beta elicits proliferation-inhibitory and differentiation-like effects in the human colon carcinoma cell line MOSER. We report for the first time that the proto-oncogene c-myc is repressed in response to transforming growth factor-beta in a human colon carcinoma cell line. We also describe a subline of these cells which are relatively resistant to the transforming growth factor-beta-induced effects on proliferation in monolayer and in soft agarose, but which retain the ability to specifically bind transforming growth factor-beta. Analysis of molecular and cellular alterations in this subline may aid in elucidating the mechanism of action of transforming growth factor-beta.     

藻蓝蛋白抑制人结肠癌SW480细胞增殖的初步研究

目的研究藻蓝蛋白对人结肠癌SW480细胞的体外抑瘤作用,为进一步探讨藻蓝蛋白抑制肿瘤的机制提供依据。方法用不同浓度的藻蓝蛋白处理结肠癌SW480细胞后,应用MTT实验来检测藻蓝蛋白对细胞增殖的抑制作用并计算出半数抑制率,利用HE染色法和电镜技术观察凋亡细胞形态结构,采用流式细胞术分析其对SW480细胞周期的影响。结果 MTT试验证明藻蓝蛋白能够抑制SW480细胞的增殖,且呈时间和剂量依赖性;HE染色以及电镜的观察结果显示藻蓝蛋白能够诱导SW480细胞的凋亡,流式细胞术显示SW480细胞被阻滞在G2/M期,G0/G1期细胞比例降低。结论藻蓝蛋白在体外能够抑制结肠癌SW480细胞的增殖,具有可开发人结肠癌治疗光敏剂应用前景。     

Spontaneous interferon production and growth of lymphoblastoid cells in serum-free medium

Numerous lymphoblastoid cell lines were established from human adult peripheral blood and cord blood lymphocytes, using Epstein Barr virus, and most cell lines from cord blood lymphocytes spontaneously produced abundant interferon without induction with Sendai virus, whereas lymphoblastoid cells from adult peripheral blood lymphocytes did not. These potential cells grow well in a newly developed serum-free culture medium based on Dulbecco's modified Eagle medium supplemented with non-essential amino acid, vitamins, nucleic acid derivatives, metal compounds, human transferrin, insulin and bovine or human serum albumin (Chon Fr.V). In serum-free medium, as well as in serum-containing conventional medium (RPMI-1640), the cells could also spontaneously produce interferon. The cells in the serum-free, culture could produce about 10 000 U/ml of interferon every day, harvesting the culture fluid and refeeding the cells with the fresh medium at the saturation cell density (10⁷ cells/ml). The interferon proved to be α-type interferon on the basis of its physico-chemical and antigenic properties.