Additional cell lines expressing both Neu and S100A4 have also been derived by transfection of rat S100A4 cDNA into tumour cell lines cloned from neu single transgenic mice. Using these cells in transfilter migration assays, it has been shown that increases in either motility or invasive properties correlate with each other and with the level of S100A4 protein. Injection of three of the cell lines separately into the mammary fat pads of nude mice showed that elevated levels of S100A4 correlated with the degree of metastasis to the lungs. In contrast, changes in cell proliferation and cell–substrate adhesion did not correlate with S100A4 levels. Neither motility nor invasiveness correlated with proteolytic degradation of gelatin as measured by zymography. Thus, the results suggest that the main effect of increases in S100A4 levels in metastasis is to generate increased cell motility and invasion and that this latter change is not dependent upon an increased ability to degrade the intercellular matrix.Elevated levels of the calcium-binding protein S100A4 are associated with poor patient survival in breast cancer patients and induce metastasis in rodent models. To investigate the effects of S100A4 on different components of the metastatic process, epithelial cells lines have been isolated from nonmalignant tumours in  The processes by which solid primary tumours, such as those of the breast, are able to disseminate and establish growth at a secondary site are still poorly understood. Tumour metastasis is complex, probably requiring both gain and loss of functions, enabling escape from the primary tumour, and growth at a secondary site. This has led to the search for metastasis-associated genes, which, unlike oncogenes, are unable to initiate tumour formation but are able to induce a metastatic phenotype in previously tumorigenic cells  foetal calf serum (FCS), 100 ng ml−1 insulin, 50 ng ml−1 hydrocortisone). The cells were tested for the expression of peanut lectin-binding protein and cytokeratin 18  (−1 Geneticin (G418). Cells were cultured in this Selective Medium for 2 weeks, the medium being replaced every 2–3 days and the resulting colonies for any one cell line pooled and expanded. Incubation of control nontransfected cells with the Selective Medium caused death of all the cells within 5 days.For transfection, cell lines grown to approximately 60% confluence in 3 cm diameter dishes were transfected with 0.5 μl of 0.125 M Tris-HCl (pH 6.8), 4% (w v−1) sodium dodecyl sulphate (SDS), 20% (v v−1) glycerol; scraped with a rubber policeman, collected in 1.5 ml tubes and protein quantitated using Bradford's reagent  following the manufacturer's instructions. Samples (10 μg) of protein from each cell line in gel loading buffer (0.125 M Tris-HCl (pH 6.8), 4% (w v−1) SDS, 20% (v v−1) glycerol, 0.04% (w v−1) bromophenol blue, 10% (v v−1) 2-mercaptoethanol), heated to 100°C, were separated by electrophoresis on 10 or 15% (w v−1) polyacrylamide gels in the presence of SDS. The separated polypeptides were transferred to nitrocellulose membranes and incubated with a primary antibody to either c-erbB-2  or S100A4 , lysed in 100 5 cells ml−1. Cell suspension (1 ml) was added to each well of a 24-well plate and at time periods of 0.5, 1, 1.5, 2.0, 2.5 h, the number of adherent cells was found by washing the wells three times with PBS to remove any cells in suspension. Any cells adhering to the well were trypsinised and counted using the Coulter counter. Each experiment was carried out in triplicate.Cells were trypsinised and counted using a Coulter counter, and resuspended at 2 × 104 cells ml−1 and 1 ml was added to each well of a 24-well plate. The cells were incubated for 24, 48, 72 h periods, and at the end of each time period the number of cells was found by trypsinising the cells and counting them in a Coulter counter. Each experiment was carried out in triplicate.Cells were trypsinised and counted as described previously, and resuspended at 1 × 10μm pores were measured using a Boyden chamber in a 24-well plate assay system . Chemotactic-induced motility in response to a FCS concentration gradient was measured by adding 400 μl of Routine Medium containing 10% (v v−1) FCS to the lower compartment, and 2 × 105 cells in 200 μl of Routine Medium, but with 2% (v v−1) FCS to the upper compartment of the Boyden chamber. The cells were incubated for 24 h, the upper side of the filter was wiped with a cotton swab to remove any nonmotile cells, and the motile cells on the lower side of the filter were fixed and stained using the Diffquik histochemical stain  according to the manufacturer's instructions. The lower compartment of the Boyden chamber was checked for cells and the number of stained cells/field of 0.50 mm2 on the lower side of the filter was counted using a Dynascope with a × 20 objective . For each cell line, four experiments were carried out, each experiment consisting of three filters and 10 fields per filter were counted. In controls, fixed numbers of cells incubated for different times up to 24 h showed a linear increase in the number of motile cells on the lower side of the filter. Thereafter, there was an appreciable number of cells present in the lower chamber.The motile and invasive abilities of the cell lines through a filter containing 8 μg Matrigel  and the experiments were carried out as for the chemotactic motility assays. The number of invasive cells/field on the lower side of the filters were determined as for the motility assays for three experiments for each cell line.For cell invasion, the filters were coated with 50 −1) FCS, 200 ng ml−1 insulin and 50 ng ml−1 hydrocortisone for 48 h. The media were harvested from each of the cell lines, grown to either 40–50% confluency (sparse cultures) or 80–90% confluency (dense cultures) and any protein concentrated into a 1 ml volume or less using an Ultrafree-15 centrifugal filter unit . The concentration of protein in each sample was then determined by measuring the absorbance at 595 nm using Bradford's reagent  following the manufacturer's instructions and relating it to the absorbance found for standards containing a known concentration of bovine serum albumin.Cell lines were washed twice with PBS and incubated in 8 ml of growth medium consisting of phenol-red-free DMEM, 2% (v v−1) SDS polyacrylamide resolving gels containing 1 mg ml−1 of gelatin, with 4% (w v−1) polyacrylamide stacking gels using the Bio-Rad Miniprotean system, as described previously (μg of conditioned medium in loading buffer (0.4 M Tris-HCl (pH 6.8), 5% (w v−1) SDS, 20% (w v−1) glycerol, 0.03% (w v−1) bromophenol blue) were loaded onto the gel, without being heated and in the absence of 2-mercaptoethanol. Gels were run at 4°C until the dye front reached the anode end of the gel. The resolving gel was washed for 2 × 30 min in 2.5% (v v−1) Triton X-100 to remove the SDS from the gel. Following which the gels were incubated for 18 h at 37°C in substrate buffer (50 mM Tris-HCl (pH 7.5), 10 mM CaCl2, 75 mM NaCl). The gel was subsequently incubated in Coomassie blue stain for 1 h and then incubated in destain (45% (v v−1) methanol, 5% (v v−1) glacial acetic acid) for 1 h, until the digested bands in the gel became clear. The gels were dried on gel drying frames between cellophane sheets .Conditioned medium was analysed on 10% . The tumours were selected from mice representative of the general phenotype of the transgene status, as demonstrated by haematoxylin and eosin-stained tumour sections. Three permanently growing cell clones were established from the mammary gland tumours of a neu transgenic mouse and termed 4Neu, 5Neu and 8Neu. Two permanently growing cell clones were established from the mammary gland tumours of a bitransgenic neu/S100A4 mouse and termed PN1 and PN2. The stable transfection of the 4Neu and 8Neu clonal cell lines isolated from the neu transgenic mouse with the rat pSV2neo-p9Ka (S100A4) expression vector yielded two further cell lines obtained from the pooled transfectant clones, termed 4Neup9Ka and 8Neup9Ka, respectively, which grew in Geneticin-containing medium. Parallel mock transfections without the expression vector yielded no colonies after incubation of cells in Geneticin-containing medium. All of the isolated cell lines were epithelial in nature as determined by staining for epithelial lineage markers such as Cytokeratin 18 and peanut lectin-binding protein (not shown)  .t-test, P<0.05). The correct sized protein of apparent molecular weight 9 kDa was seen for all cell lines . The correct sized protein of 185 kDa was seen for all the cell lines, but an additional band at 135 kDa was sometimes seen; this may have been due to crossreactivity with the EGF receptor   protein levels    , althoug data in .t-test, P<0.03)  were higher than those isolated from neu transgenic mice   . Moreoveu, 8Neu) .t-test, P<0.05). When S100A4 protein levels are plotted against motility, there is a significant linear relationship when assessed by a least-squares regression analysis   (Comparison of S100A4 levels with cell motility for the different cell lines showed that S100A4 levels and motility were closely associated . This diP=0.012) . In the  P=0.88) .t-test, P<0.03)  were higher than those isolated from neu transgenic , that for PN2 was significantly so .To allow for direct comparison between cell invasion and cell motility, the invasive abilities of the cell lines were measured using the assay conditions optimised for the motility assay but with a coating of Matrigel on the filter separating the upper and lower compartment of the Boyden chamber. Using this assay, cell invasion for the different cell lines varied from about three to nearly 26 invading cells per field . The inv P<0.03) . Moreovet-test, P<0.01) and demonstrated a significantly higher invasive ability when compared individually to 8Neu . A significant linear correlation between S100A4 protein levels and invasive ability was demonstrated by least-squares regression analysis for each cell line    . However P=0.96) .R2=0.95; P=0.00096)  . Interes0.00096) . It woul−1) FCS, the same medium as that used in the upper chamber of the Boyden system during the invasion assay. To analyse any effect of cell confluency on MMP production, medium was harvested from sparse cultures of cells, grown to 40–50% confluency and from dense cultures of cells grown to 80–90% confluency.Matrix metalloproteinase (MMP) activities were measured by gelatin zymography of conditioned media harvested from cell lines grown in Routine Medium supplemented with 2%  and MMP9 (Mr 78 kDa)  (P<0.03). The incidence of metastasis in the high S100A4-expressing PN2 cell line was also greater than that in the moderate S100A4-expressing 8Neup9Ka cell line, although not statistically significant . Furthermore, when the level of S100A4 protein in the cultured cells was plotted against the percentage of tumour-bearing mice with lung metastases, a straight line could be drawn through the three points  . Metastases were also observed in the inguinal lymph nodes of some of the mice injected with the 8Neup9Ka and PN2 cell lines; however, these were not included in any statistical analyses, since lymph node tissue was not available from all of the injected mice.The primary tumours consisted of chord-like structures  . In contImmunocytochemical staining for Neu showed that all three cell lines 8Neu (Figure 5A and PN2  produced and PN2 . Lymph n and PN2 , the mic and PN2  and the  and PN2  in the l and PN2 . The tum and PN2 , which v and PN2 . Immunoc and PN2 , and all and PN2 . There w and PN2 . Althoug and PN2 . These rneu transgene or by isolation of cell lines from mice carrying both the neu and the S100A4 transgenes. The former mice produce only nonmetastatic tumours, while the latter mice produce metastatic tumours (in vitro, which may be related to metastasis in vivo. There was no correlation between the levels of S100A4 and cell-substrate adhesion to plastic substrata or proliferation rates for this particular set of cell lines; cell–cell adhesion was not measured. These data are the first to demonstrate in a transgenic murine-derived tumour system that S100A4 levels correlate positively in a linear fashion with cell invasive ability through Matrigel in response to an FCS density gradient. This result demonstrates also that S100A4 causes, either directly or indirectly, the enhanced invasive ability in at least the S100A4-transfected cell lines. In contrast, correlation of levels of Neu protein with only proliferation rates and not with substrate adhesion, motility or invasion is consistent with the expression of the neu transgene causing the largely proliferative breast lesions and noninvasive breast tumours observed in transgenic mice (neu transgenic mouse tumour. These analyses still yielded the same significant correlations as before (Cell lines expressing elevated levels of S100A4 have been obtained either by direct transfection of cell lines from mice carrying the nic mice , the anas before .in vitro of Lewis lung carcinoma cell lines (in vitro (Whether production of S100A4 correlates with invasive ability has remained equivocal. Previously, S100A4 had been shown to correlate with the invasive ability in vivo is more complex (Matrix metalloproteinase production could, however, be regulated in all of the cell lines by confluency in culture. Matrix metalloproteinase activity was observed only in the conditioned medium of sparsely plated cells. It would therefore seem that complete cell–cell contact provides an inhibitory extracellular signal, which prevents MMP production/secretion in these cells. Previous studies have also demonstrated that proteolytic activity is dependent on cell density. For example, in the media of sparse astrocytoma cells, more collagenase activity was seen than in dense cultures (in vitro, showed a strong linear correlation with levels of S100A4 protein in the different cell lines. The stimulus for motility did not need to be directional, since chemotactic and chemokinetic stimuli provided by 10% FCS, induced the same degree of motility (in vitro suggest that an appropriate and sufficient chemical stimulus is probably required for cell migration in vivo. The results demonstrated here are in accordance with previous findings in other cell systems that increased S100A4 levels results in increased cell motility (in vitro, were not invasive in vitro or in vivo, due to a lack of MMP production (In contrast to gelatinase activity, motility, a second major component of invasion examined motility . Howevermotility , suggestmotility  suggestsmotility , the thrin vitro (inter alia (The mechanisms whereby S100A4 modulates cell motility are unknown. However, S100A4 is known to bind to various components of the actin–myosin cytoskeleton (neu and neu/S100A4 transgenic mice increased the efficiency of mammary tumour production from 44 and 68% as seen after 14 months in neu and neu/S100A4 transgenic mice (neu was observed in the cell lines. There was no requirement to add hormones in vitro or to mate in vivo to stimulate the expression of neu, whereas in the original transgenic mice the neu transgene requires the hormones of pregnancy to activate its MMTV promoter (neu transgenic mice all of the epithelial cells express the neu transgene, but only some cells are able to form tumours. This latter result indicates that a second genetic change is required, in addition to expression of the activated neu transgene, to allow tumour formation. One possible explanation is the high level of the loss of heterozygosity, a fact which may be related to the loss of tumour suppressor genes, which has previously been observed in these neu transgenic mice (The tumorigenic and metastatic abilities of the cell lines were examined in the immunoincompetent, nude mouse, without the complication of an immune response. The isolation and culture of tumour cells from both nic mice . Since cneu/S100A4 tumour-derived PN2 cell line as in the neu/S100A4 transgenic mice, with only 50% of mice with mammary gland tumours developing lung metastases. The efficiency of tumour metastasis for the 8Neup9Ka cell line was lower, with only 30% of mice with mammary gland tumours developing lung metastases. The 8Neu cell line developed no lung metastases. These in vivo results follow the same pattern observed for the cell lines in in vitro assays, where the 8Neu cell line had a low level, the 8Neup9Ka cell line a moderate level, and the PN2 cell line a high level of both motility and invasion. Confirmation that these metastases are derived from the neu/S100A4-expressing mammary gland tumours is obtained by positive staining of these metastases for both Neu and S100A4. The level of metastasis expressed as a percentage of afflicted animals was found to be positively correlated with the level of S100A4, but not with the level of Neu protein in the cell lines, albeit for only three of the cell lines. Metastasis in these Neu-expressing cell lines is therefore clearly dependent, either directly or indirectly, on the level of S100A4 protein.The frequency of tumour metastasis, however, remained the same in the neu acts as an oncogene producing neoplastic transformation of the targeted mammary epithelial cells in vivo (neu these cells remain normal and cannot be grown in culture (neu transgenic mice (in vivo. Tissues of transgenic mice overexpressing S100A4, on the other hand, fail to undergo neoplastic transformation and that includes those of the mammary gland (neu-transformed neoplastic immortalised mammary cell lines, in vitro studies demonstrate that S100A4 levels correlate well with cell invasion and that S100A4 mediates, either directly or indirectly, this invasion through its ability to induce cell motility, at least in the transfected cells. It therefore seems probable that S100A4-driven cell motility mediates invasion in vivo and thereby metastasis in vivo, at least in this particular model system.In this transgenic mouse model system,  culture  without


Some tumour cell lines, notably the vast majority of melanomas and hepatocellular carcinomas, tend to be deficient in argininosuccinate synthetase (EC 6.5.4.3.), and therefore cannot recycle citrulline to arginine. Argininosuccinate synthetase is present at levels that convert enough citrulline to arginine to allow limited growth in about half of a modest range of malignant cell types analysed in this study. Attempts to rescue cells that are unable to utilise citrulline with the immediate downstream product, argininosuccinate, had very limited success in a few tumour cell lines. Particularly noteworthy is the demonstration that argininosuccinate was totally incapable of rescuing cells that utilise citrulline efficiently, consistent with tight channelling (coupling) of argininosuccinate synthetase and argininosuccinate lyase in the urea cycle. The findings suggest that an excellent opportunity exists for further exploitation of arginine deprivation in the selective killing of tumour cells.Arginine deprivation causes death of up to 80% of cancer cell lines  Having successfully reduced L1210 cells in culture to negligible viability in 3–5 days with arginase (EC 3.5.3.1), citrulline supplementation in the continued presence of the enzyme partially circumvented the arginine deficiency. The rate of conversion became a rate-limiting factor, most noticeably in such fast-growing leukaemia as L1210 . In a corresponding in vivo study, arginase treatment of DBA/2 mice injected intraperitoneally with 1210 cells increased neither their survival time nor decreased tumour burden, despite plasma arginine falling to ∼1–2 μM level. Plasma citrulline remained normal, and therefore the tumour cells must have converted citrulline to arginine fast enough to sustain relatively normal tumour growth rate  generation). Its removal from culture medium by medium formulation or arginase treatment quickly leads to death in ∼80% of tumour cell lines . Counts are representative of the normal cell population, within standard parameters, and unless otherwise stated cannot be taken as an indication of viability. All cell cultures were routinely screened, and just prior to experimental use, for four types of mycoplasma, using the Mycoplasma Screening Kit of Roche , according to the manufacturer's instructions to ensure that no cultures used for experimental work tested positive.Cells from a wide range of suppliers and sources were grown in RPMI 1640 medium  at 37°C in a humidified atmosphere of 5% COM arginine and 5% dialysed foetal calf serum (FCS) overnight. Prior to treatment, cells were washed with prewarmed arginine-free medium (hereafter AFM) and treated with fresh AFM to which arginine, argininosuccinate, citrulline and ornithine was added at 1 mM, where appropriate. Native bovine arginase  or human recombinant arginase  was added at a concentration of 1 U ml−1 to arginine-containing RPMI. In the case of L1210 cells, a suspension culture line, cells were treated immediately on being resuspended at the desired concentration in AFM to which arginine, argininosuccinate, citrulline and ornithine were added as desired.Cells were seeded at approximately 50 000 cells per well in 12-well plates and allowed to settle in RPMI medium containing 1 min vitro overtime at 37°C in a humidified atmosphere of 5% CO2 to show that it maintained activity over the course of each experiment, using the ornithine method of μmol min−1 mg protein−1. In all, 1 U of arginase was taken as the amount that broke down 1 μmol arginine min−1.Arginase was analysed  pathway  because The findings on human melanomas contrast sharply with those on mouse B16F10 melanoma cells, which efficiently handle citrulline , but unlall tumour cell types given in in situ from citrulline) is used by the lyase to form arginine. The three human leukaemic cell lines  arginine. Although rescue with argininsuccinate in AFM with traces of contaminating arginine is therefore very unlikely, it would have been pre-emptorily destroyed before the cells could make use of it where arginase had been used to create the deficiency  normally operates, thereby preventing the access of argininosuccinate to argininosuccinate lyase (EC 4.3.2.1.), which would explain the absence of Type C/AS tumours . This paRescue of cell lines incapable of utilising citrulline with argininosuccinate does occur, but with considerable differences between the cell lines. CHO is an excellent example of a cell line that cannot use it and is therefore totally dependent on arginine. This is more noteworthy because the generalisation of not exclusively show this argininosuccinate synthetase deficiency, the findings show that most cases of this dangerous and aggressive tumour can be appropriately and quickly selected for treatment. They are the preferred targets for therapy, for which screening of primary tumours has already begun.Nevertheless, the inability of a number of tumour cell lines to utilise citrulline renders them prime targets for eradication by arginine deprivation, whether achieved by leaving it out in medium formulation or using catabolic enzymes in its presence. This potential Achilles' heel in tumour metabolism deserves further exploitation in view of our previous findings (


Concentrations of methylprednisolone which have lethal effects on human lymphoblastoid cell lines in vitro cause a reduction both in the uptake of uridine and thymidine into acid-soluble material and their incorporation into acid-insoluble material. These effects are virtually instantaneous, which indicates that they do not depend on alterations in gene activity. Normal uptake of nucleosides into cells is by both simple and facilitated diffusion, and methylprednisolone appears to act directly on the cell surface to inhibit only facilitated diffusion uptake.


The expression of the gp100 antigen is generally thought to be confined to cells of the melanocytic lineage, which makes the protein a suitable melanoma-specific marker. Strikingly, after screening a panel of normal tissues, tumour samples and cell lines of non-melanocytic origin, we found transcripts encoding gp100 in virtually every tissue and cell line tested. In contrast, tyrosinase and MART-1/MelanA transcripts were detected only in cells of the melanocytic lineage. However, no gp100 protein could be detected by either Western blotting or cytotoxicity assays. Therefore, at the protein level, gp100 remains exclusive for cells of melanocytic origin despite its transcription in many cell types. The major implication of this finding is that screening of patient material for gp100 expression should preferrably be performed by antibody staining. Reverse transcriptase polymerase chain reaction (RT-PCR) can be employed, provided that it is performed in a tightly controlled, semiquantitative setting.


The Wnts can be classified into two classes based on their ability to transform cells. The Wnt5a class can antagonize the effects of transforming Wnts partly through effects on cell migration. To understand the mechanisms of regulation of Wnt5a, we investigated its expression in human normal and breast cancer cell lines. Elevation of Wnt5a in HB2, a normal breast epithelial cell line, was linearly correlated with cell density, but this did not occur in cancer cell lines. We examined intracellular events responsible for the regulation of Wnt5a by cell to cell contacts, using various metabolic agents known to affect signal transduction pathways. Agents that selectively blocked protein kinase C  or protein tyrosine kinases (genistein) reduced the level of Wnt5a expression markedly. Protein kinase C activation by phorbol 12-myristate 13-acetate up-regulated Wnt5a partly through prolongation of Wnt5a mRNA half-life. Cytoskeleton reorganization following cytochalasin D treatment caused an induction of Wnt5a, which was associated with changes in cell morphology. Calphostin C did not block these effects, showing that protein kinase C is acting upstream of cytoskeletal modulation. However, the cancer cell lines treated with cytochalasin D showed no changes in cell morphology or Wnt5a induction, suggesting disruption of this regulatory pathway in cancer.


Bryostatin 1 is a novel macrocyclic lactone activator of protein kinase C (PKC) which has clinical potential as an anti-cancer agent. The mechanism of action of this agent is unknown, but protein kinase C has been implicated. In order to investigate this possibility, we have developed P388 sublines resistant to bryostatin 1, by continuous challenge of the parent cell line with increasing incremental concentrations of the drug over 4 months. Cell lines were established at monthly intervals yielding four sublines: P388/BR/A, which were removed at 1 month; P388/BR/B, obtained after 2 months; P388/BR/C, obtained after 3 months; and P388/BR/D, which were established after 4 months. All four P388/BR sublines show an equal degree of resistance to the growth inhibitory effects of bryostatin 1, with a relative resistance ratio (RR) IC50 of approximately 4,000. The ability of the cytosol of cells to phosphorylate PKC-specific substrate is decreased by 41% for BR/A, 57% for BR/B 80% for BR/C and 94% for BR/D compared with the parental cell line, even when grown in the absence of bryostatin 1 for up to 4 weeks. Similar decreases are seen for cytosolic phorbol ester binding and whole-cell PKC isoenzyme expression. All four P388/BR sublines show high and equal levels of cross-resistance to the PKC activatory phorbol ester, phorbol 12-myristate 13-acetate (PMA). There is no loss of resistance to either bryostatin 1 or PMA up to 3 months after termination of exposure of the sublines to bryostatin 1. There was no significant degree of cross-resistance to daunorubicin in the bryosatin 1-resistant cell lines, P388/BR/A, B, C or D, when compared with the parent cell line, P388.


SP-G inhibits mitogenic neuropeptide signal transduction and small cell lung cancer (SCLC) cell growth in vitro and in vivo. Using the SCLC cell line series GLC14, 16 and 19, derived from a single patient during the clinical course of their disease and the development of chemoresistance, it is shown that there was an increase in responsiveness to neuropeptides. This was paralleled by an increased sensitivity to SP-G. In a selected panel of tumour cell lines , the expression of the mitogenic neuropeptide receptors for vasopressin, gastrin-releasing peptide (GRP), bradykinin and gastrin was examined, and their sensitivity to SP-G tested in vitro and in vivo. The tumour cell lines displayed a range of sensitivity to SP-G . The expression of the GRP receptor measured by reverse transcriptase–polymerase chain reaction, correlated significantly with growth inhibition by SP-G. Moreover, introduction of the GRP receptor into rat-1A fibroblasts markedly increased their sensitivity to SP-G. The measurement of receptor expression from biopsy samples by polymerase chain reaction could provide a suitable diagnostic test to predict efficacy to SP-G clinically. This strategy would be of potential benefit in neuropeptide receptor-expressing tumours in addition to SCLC, and in tumours that are relatively resistant to conventional chemotherapy.[Arg Despite recent advances in cytotoxic drug development, it is a universal goal to develop novel cancer therapies that are more specific to cancer cells and produce minimal damage to nonmalignant cells. One approach to realising this goal is the development of drugs that are able to block the growth-promoting effects of cancer cell mitogens.in vitro and also as xenografts in nude mice  (SP-G), were initially identified as antagonists of substance P-mediated cellular effects and were subsequently found to also antagonise the cellular effects of bombesin . SCLC cells proliferate in response to a range of neuropeptide growth factors, and in many cases these neuropeptides are involved in autocrine and paracrine growth loops that fuel unrestrained proliferation , gastrin-releasing peptide (GRP), bradykinin (BK2) and gastrin by reverse transcriptase–polymerase chain reaction (RT–PCR), and show that GRP-receptor expression predicts sensitivity to SP-G in a variety of tumours , colorectal, ovarian and pancreatic) in vitro and in vivo. Our results suggest that an antineuropeptide growth factor strategy may be effective in a wider range of tumours and may also be of benefit in these neuropeptide-expressing tumours, which have acquired relative resistance to conventional chemotherapeutic agents.In this study, we measured the expression of the mitogenic neuropeptide receptors for vasopressin (VRat-1a cells and rat-1a cells stably expressing the mouse bombesin/GRP receptor (GRPR) (BOR-15) were established by the Imperial Cancer Research Fund . RPMI-1640, Dulbecco's essential Eagle's medium (DMEM), bombesin and vasopressin were from Sigma . SP-G was a kind gift from Peptec . All other reagents were of the purest grade available.−1) fetal bovine serum (heat-inactivated at 57°C for 1 h) 50 U ml−1 penicillin, 50 μg ml−1 streptomycin and 5 μg ml−1L-glutamine in a humidified atmosphere of 5% CO2 : 95% air at 37°C. For experimental purposes, cells were either grown in SITA medium ) or with 0.1% (v v−1) fetal bovine serum in DMEM (nontumour cell lines). Rat-1A fibroblasts stably transfected with the mammalian bombesin receptor (BOR-15 cells) were cultured in the presence of 400 μg ml−1 G418-sulphate.Stocks were maintained in RPMI-1640 (tumour cell lines) or DMEM (nontumour cell lines) supplemented with 10% . Colony growth was determined in 0.3% agarose in SITA medium for 21 days as described by 2+concentration was determined using the fluorescent indicator Fura-2-tetraacetoxymethylester AME (FURA-2-AM 1 μM) as described (Intracellular Canu/nu mice (6 weeks old)  were maintained in negative pressure isolators . All xenografts used throughout were initially established by subcutaneous injection of 107 cells from their respective cell lines and then maintained by serial passage of fragments of viable tumour as described previously  was then calculated by the formula V=π/6LW2, where L is the longest diameter and W the diameter perpendicular to L. Results were expressed as a relative tumour volume (RTV) which is defi-ned simply as: V on day x/V on day 0, with RTV=1 on day 0. Xeno-graft experiments were repeated on two or three separate occasions.Mice were allocated into control and treatment groups containing six to eight mice. Treatment started when the xenografts reached a diameter of 3–10 mm with the first day of peptide administration designated as day 0. SP-G was dissolved in sterile distilled water at a concentration of 10 mg ml1AR sense 5′-TACCTGCTACGGCTTCATCTGC-3′, antisense 5′-ACACAGTCTTGAAGGAGATGGC-3′; BK2R sense 5′-CCTGAGTGTCATCACCTTCTGC-3′, antisense 5′-TTGATGACACGGCAGAGG-3′; receptor for gastrin (gastrinR) sense 5′-CCTATCTCCTTCATTCACTTGC-3′, antisense 5′-AGTGTGCTGATGGTGGTGTAGC-3′; γ-actin sense 5′-ATGGCATCGTCACCAACTGG-3′, antisense 5′-ATGACAATGCCAGTG-GTGGC-3′. Polymerase chain reaction products were run on a 1.5% agarose gel containing 1 μg ml−1 ethidium bromide. Stored images of the gels were analysed by densitometry using Gel Base/Gel Blot software. γ-Actin levels were measured as an internal control. Each product was expressed relative to the levels of γ-actin for the same cDNA batch and for each PCR reaction.RNA was extracted using Tri-Reagent  from exponentially growing cells, which had been in SITA medium for 3–5 days and was subsequently treated with DNase1 to remove any DNA contamination. cDNA was produced from this RNA using a first-strand synthesis kit . PCR primers used were: GRPR sense 5′-ATCTTCTGTACAGTCAAGTCC-3′, antisense 5′-GCTTTCCTCATGGAAGGGATA-3′; Vin vitro sensitivity to chemotherapeutic agents reflected the clinical resistance to treatment (M) in each of the three cell lines were screened for their ability to increase [Ca2+]i. 2+]i and smaller responses were seen in case of neuropeptides CCK, GRP and neuromedin B. No Ca2+ mobilisation was observed in response to neurotensin, substance P or vasopressin. However, in the GLC19 line, addition of neuropeptides bradykinin, CCK, GRP, neuromedin B and substance P caused large increases in [Ca2+]i. Vasopressin and neurotensin caused smaller but consistent increases in [Ca2+]. The GLC19 was the only cell line in which neurotensin caused a measurable increase in [Ca2+]i.The GLC14, 16 and 19 cell lines are SCLC cell lines established from one patient during longitudinal follow-up  and the HT29 colorectal cell line (IC50=18 μM). Four out of five SCLC cell lines had IC50 values below the mean IC50 value of 44 μM. Both the ovarian carcinoma and the non-small-cell carcinoma were sensitive to growth inhibition by SP-G, with IC50 values of 31 and 33.5 μM, respectively. During the phase 1 clinical trial of SP-G, plasma levels of up to 40 μM were achievable with no dose-limiting toxicity , NSCLC and colon cancer, which are relatively insensitive to standard conventional chemotherapy.Four members from the original panel of tumour cell lines were established as xenografts from their respective cell lines: H69 SCLC, HT29 colon carcinoma, PEO4 ovarian cancer and PANC-1 pancreatic cancer. Typical growth inhibition curves . Receptors for vasopressin (V1AR) were detected in four out of four lung cancer cell lines (three SCLC and one NSCLC). The WX330 SCLC cell line was the highest expresser of V1AR. The pancreatic and ovarian cancer lines expressed low levels of the V1AR and in the colorectal cancer cell lines only HT29 cells expressed V1AR. GRPRs were detected in three out of four lung cancer cell lines (two SCLC and one NSCLC). In the colorectal cell lines, the HT29 cell line showed high levels of GRPR expression and HCT116 cell line showed no detectable GRPR expression with intermediate expression in the HT18 cell line. The pancreatic cell line PANC1 had very low levels of GRPR expression. The PEO4 ovarian cancer cell line and H69 SCLC cell lines showed high expression of the GRPR (γ-actin) and sensitivity to SP-G was the most apparent  , whereas BOR15 cell growth was completely inhibited by 80 μM SP-G  was expressed at high levels . The sensitive cell lines  had IC50 values that were in the range of physiologically obtainable plasma concentrations. Moreover, in the cell lines tested in vitro sensitivity correlated with sensitivity to SP-G in vivo. The results of this screen suggest that SP-G could be used therapeutically against several tumour types in addition to SCLC.It is well known that neuropeptides can stimulate the growth of many types of cancers including pancreatic, colorectal, prostate, ovarian, breast and NSCLC (in vitro (and in vivo) are resistant to standard chemotherapeutic agents such as etoposide, are sensitive to the growth-inhibitory effects of SP-G. This raises the possibility that SP-G may be an effective anticancer agent in patients with neuropeptide-expressing tumours which are intrinsically resistant or have acquired resistance to conventional chemotherapy. A plausible extension of this hypothesis is that clinically aggressive drug-resistant SCLC cells that emerge after chemotherapy might have a more extensive network of neuropeptide regulation and therefore display increased sensitivity to neuropeptide antagonists. Further studies in longitudinal cell lines are required to investigate this hypothesis in further detail.We have previously shown that SP-G augments etoposide-induced growth inhibition and apoptosis in SCLC cells and suggested that SP-G may be of increased benefit in patients following relapse or in conjunction with conventional chemotherapy  pathways leading to growth arrest and apoptosis (The GRPR has been shown to be oncogenic when transfected into the nonmalignant NCM460 colon epithelial cell line (These findings have important implications for the design of more advanced phase human clinical trials using substance P analogues. SP-G is currently entering a phase II clinical trial where its effectiveness will be tested in SCLC patients, but ultimately, compounds of this type may also be suitable for the treatment of a wide range of other tumour types and neuropeptide-expressing tumours that have become resistant to standard conventional chemotherapeutic agents. Screening tumour biopsy samples for neuropeptide receptor expression may provide insight into the likelihood of patients responding to treatment with substance P analogues, analogous to oestrogen receptor expression conferring efficacy to tamoxifen in breast cancer. It is suggested that tumours should be biopsied to select patients for substance P-analogue trials based on the expression of the GRPR and another neuropeptide receptor. Our results suggest that these tumours should show growth inhibition regardless of intrinsic or acquired resistance to standard chemotherapeutic agents. It is therefore proposed that in the first instance, patients with tumours that express the GRPR and another neuropeptide receptor who have failed conventional treatment should be randomised into two groups – best supportive care and treatment with substance P analogues.


A cytotoxicity assay was used to study the action of bacillus Calmette-Guerin (BCG) and cytokines on four human bladder cancer cell lines. Monocytes and lymphocytes from peripheral blood were incubated with or without BCG or cytokines for 24 h, after which [3H]thymidine-labelled target cells were added and the 72 h percentage specific release determined. BCG had a direct cytotoxic effect against tumour cells and significantly enhanced monocyte/macrophage and enhanced lymphocyte cytotoxicity against one cell line (UCRU-BL-17). Supernatants (SNs) from BCG-activated monocytes/macrophages and lymphocytes increased the percentage specific release of [3H]thymidine from UCRU-BL-17 cells. Interferon alpha  and interleukin 2 (IL-2) were cytotoxic towards UCRU-BL-17. No synergy occurred between BCG and cytokines at the concentrations tested. The results suggest that BCG is superior to IFN-alpha, interferon gamma (IFN-gamma) and IL-2 in enhancing cell-mediated cytotoxicity.


Thymidine (1 μM) completely prevented growth inhibition at the MTA IC50in all cell lines. At 10 × IC50, growth inhibition was only partially reversed by thymidine (≤ 10 μM); both thymidine and hypoxanthine (30 μM) being required for complete reversal, reflecting the multi-targeted nature of MTA. Growth inhibition by MTA was not affected by hypoxanthine alone. A non-toxic concentration (1 μM) of DP prevented thymidine/hypoxanthine rescue of MTA indicating that DP may potentiate MTA activity by preventing nucleoside and/or base salvage. Thymidine transport was inhibited by ≥ 89% by 1 μM DP in all cell lines, whereas hypoxanthine transport was inhibited only in A549 and MCF7 cells. Therefore, prevention of end-product reversal of MTA-induced growth inhibition by DP can be explained by inhibition of thymidine transport alone. © 2000 Cancer Research CampaignThe novel pyrrolopyrimidine-based antifolate LY231514 (MTA), inhibits multiple folate-requiring enzymes including thymidylate synthase, glycinamide ribonucleotide formyltransferase and dihydrofolate reductase. Both thymidine and hypoxanthine are required to reverse MTA growth inhibition in leukaemia and colon cancer cells. Prevention of MTA growth inhibition by thymidine and/or hypoxanthine was investigated in two human lung  and two breast  tumour cell lines, and the effect of the nucleoside/base transport inhibitor dipyridamole (DP) on thymidine and hypoxanthine rescue defined. MTA IC


The development of resistance to cisplatin (DDP) occurs rapidly both in vitro and in vivo, and constitutes a major obstacle to effective therapy. We have previously demonstrated that there is a highly synergistic interaction between tamoxifen (TAM) and DDP against cell lines representative of three different human cancers: melanoma, ovarian carcinoma and small-cell lung cancer. The purpose of these studies was to determine if TAM interferes with the development of resistance to DDP. T-289 melanoma cells and 2008 ovarian cancer cells were cultured with increasing concentrations of DDP +/- TAM in an attempt to induce resistance to DDP. At various time points the cells were removed from culture and the degree of resistance to DDP was quantitated. Concurrent exposure to TAM and DDP decreased both the rate and the absolute magnitude of resistance to DDP in both melanoma and ovarian cancer cell lines. In the T-289 cell line the rate was decreased by a factor of 3.4 +/- 1.4 (P < 0.05), while in the 2008 cell line the rate was decreased by a factor of 2.4 (P < 0.01). TAM decreases the rate as well as the absolute magnitude of in vitro resistance to DDP in both melanoma and ovarian cancer cell lines. These data suggest that the concurrent administration of TAM and DDP may result in a delay in the development of resistance to DDP which may have important clinical implications in the design of DDP-containing regimens.


The hydro-alcoholic extracts of the plants were prepared by percolation. The cytotoxic effects and IC50 of the extracts on the cell lines were studied followed by colonogenic assay after 72 h incubation. The results showed that the extract of Juniperus sabina possesses lower IC50 in comparison with Zataria multiflora extract on all 4 normal and cancer cell lines (P<0.05); but, IC50 of the Juniperus sabina extract was significantly higher than the Taxus baccata extract and Cisplatin on all 4 normal and cancer cell lines (P<0.05). As a result, it is concluded that the extract of J. sabina has almost similar cytotoxicity with the extract of Taxus baccata on cancer cells.Isolation and identification of some potent anti-tumor compounds from medicinal plants has motivated researchers to screen different parts of plant species for the determination of anti-tumor effects. In this study, cytotoxic effects and IC Isolation and identification of some potent anti-tumor compounds, such as colchicine, Vinca alkaloids, and also docetaxol and paclitaxel (taxol), as one of the most consumed natural anticancer compounds have encouraged scientists to study the toxico-pharmacological effects of different parts of plant species against cancer cell lines.–8Juniperus sabina possess cytotoxic effects on some human cancer cell lines.[Juniperus sabina  showed the presence of cytotoxic compounds or tumor necrotizing substances.[Taxus species. However, difficulty of obtaining this compound from yew trees has limited its clinical use.[It has been previously reported that all parts of the yew plant contain poisonous taxine alkaloids. Taxines save their toxic effects during the year, with itsll lines. The potebstances.–2 Taxol ical use.Zataria multiflora Boiss belongs to the family Laminaceae that geographically grows in Iran, Pakistan and Afqanistan.[Z. multiflora showed inhibitory effects on Salmonella typhimurium and Staphylococcus aureus in brain heart infusion (BHI) broth medium.[qanistan.14 This pqanistan. In a stuh medium.Cisplatin (cis-dichlorodiammineplatinum-II) gained a widespread use against various malignant tumors in experimental animals18 and va20222650 of specific concentrations of hydro-alcoholic extracts of fruits of Juniperus sabina and leaves of Zataria multiflora were compared with hydro-alcoholic extract of bark of Taxus baccata and Cisplatin, as well as known herbal and chemical anticancer compounds on normal (CHO and rat fibroblast) and cancer (HepG2 and SKOV3) cell lines.In this investigation, cytotoxic effects and ICJ. Sabina and bark of T. baccata were collected from the northern regions of the Iran  in September 2007. Also, leaves of Z. multiflora were collected from downtown of Shiraz (Fars province). The plant specimen was identified by the Department of Pharmacognosy, Sari faculty of Pharmacy, and stored at −20°C.Fruits of A measured quantity of 50 g of dried and powdered parts of each plant was chopped and soaked in 75 ml of ethanol (80% v/v) for 24 h and then percolated . The extrCHO , normal rat fibroblast, HepG2 (Human hepatocarcinoma) and SKOV3 (Human ovary carcinoma) cell lines were purchased from Pasture Institute .The completed media were sterilized by 0.22 μ microbiological filters and kept at 4°C before use.Juniperus Sabina, Zataria multiflora and Taxus baccata, and 50 μl of 0, 2.5, 5, 10, 25 μg/ml of Cisplatin for 2 h; and then washed using sterile normal saline 0.09%.In Colonogenic assay, 50 μl of DMEM/F12 including 500-700 cells were added to 3 wells of 6well/plates for each concentration of the extracts and Cisplatin. Then, they were incubated for 48 h. After incubation, the cell lines were exposed to 50 μl of 0 (phosphate buffer), 5, 25, 50, 100, 150 μg/ml of hydro-alcoholic extracts of Then, 4 ml of fresh culture media was added to the wells, and incubated for seven days. After this period, the contents of wells were excluded; the cells were fixed by formalin 9%, and dyed by trypan blue 4% (w/v) for 20 min. Then, trypan blue was excluded, and the six well/plates were washed using sterile normal saline 0.09%. At the end, the dyed colonies were counted by light microscope.P<0.05).Prism ver.3 Software was used to perform statistical analysis. One-way ANOVA method followed by Tukey test was used to determine the differences among the groups . But, IC50 of the Juniperus sabina extract was significantly higher than the extract of Taxus baccata and Cisplatin on all 4 normal and cancer cell lines (P<0.05) [50 represent the higher potency of a compound to inhibit the growth of cells and cause toxicity and death of cells.The results showed that the extract of (P<0.05) . The low50 of the Juniperus Sabina and Zataria multiflora extracts with the Taxus baccata extract and Cisplatin on normal and cancer cell lines are showed at 50 was related to cisplatin and hydro-alcoholic extract of Zataria multiflora in all cell lines. IC50 of the compounds on the 4 cell lines increased according to the rank order of cells Cisplatin < Taxus baccata < Juniperus sabina < Zataria multiflora.Comparison of the evaluated IC50 = 0.87±0.07 μg/ml) and CHO cell lines (IC50 = 5.5±0.21 μg/ml).On the other hand, the highest and lowest cytotoxicity of Cisplatin was related to HepG2 (IC50 of cisplatin on the 4 cell lines increased according to the rank order of cells CHO > Fibroblast > SKOV3 > HepG2.The IC50 of hydro-alcoholic extract of Taxus baccata on the 4 cell lines increased according to the rank order of cells CHO > Fibroblast > HepG2 > SKOV3.On the other hand, the IC50 of hydro-alcoholic extract of Juniperus sabina on the 4 cell lines decreased according to the rank order of cells CHO < Fibroblast < HepG2 < SKOV3.Also, the IC50 of hydro-alcoholic extract of Zataria multiflora on the 4 cell lines decreased according to the rank order of cells Fibroblast < CHO < HepG2 < SKOV3.The IC50 of the Juniperus sabina and Zataria multiflora extracts, and Cisplatin, as drug control positive compound and Taxus baccata extract, as plant control positive on normal cell lines were higher than that on cancer cell lines. This difference can be resulted from dysfunction of cellular organisms following cancer incidence which cause higher rate of proliferation and increased cellular intake. Also, defensive disorders and effusion insufficiency to escape toxic substances from cells can lead to lower necessity to amounts of cytotoxic compounds to inhibit the growth of cancer cells in comparison with normal cells.[According to the results, ICal cells.8Juniperus sabina and its insignificant differences with IC50 of the extract of Taxus baccata, as a common natural anticancer product, allows us to conclude that extracts of different parts of Juniperus sabina are good candidates for further studies of activity-monitored fractionation to identify their active components.Determination of viability percent of the cells treated with the extract of Juniperus Sabina and Zataria multiflora on cancer cell lines and compared their IC50 with the hydroalcoholic extract of Taxus baccata and Cisplatin. We have observed that Juniperus sabina has insignificant differences with IC50 of the extract of Taxus baccata on HepG2 and SKOV3 cancer cells. This result presented another novel approach for the treatment ofsome cancers.In this study, we have determined the cytotoxic activity of hydroalcoholic extracts of


Poly(ADP-ribose) polymerase (PADPRP), which uses NAD to synthesize ADP-ribose polymers, is activated by DNA strand breaks and mediates cellular responses to DNA damage. The consequences of low cellular NAD levels in a cell line deficient in nicotinamide mononucleotide adenylyltransferase (NMNAT), an enzyme essential for NAD biosynthesis, were investigated by assessing NAD metabolism and DNA repair after treatment with alkylating agents. A tiazofurin-resistant L1210 cell line (TZR) was isolated. NAD levels were approximately 5933 and 3375 pmol mg(-1) protein for parental  and TZR cells respectively, and NMNAT levels were reduced by > 95%. TZR cells were more sensitive to temozolomide (TM) and 1-methyl-3-nitro-1-nitroso-guanidine (MNNG), particularly at concentrations that caused > 50% NAD depletion. TM and MNNG treatment decreased NAD levels in both cell lines, but took longer to return to control levels in TZR cells. For example, MNNG (5 microM), depleted NAD levels at 6 h to approximately 4512 (WT) and 1442 (TZR) pmol mg(-1) protein; however, NAD levels had returned to control levels by 8 h in WT cells, but were not restored by 16 h in TZR cells. Both cell lines were equisensitive to the growth-inhibitory effects of NU1025 per se (IC50 370 microM). Co-exposure of the cell lines to TM (100 microM) with increasing concentrations of NU1025 led to a synergistic enhancement of cytotoxicity, with IC50 values for NU1025 decreasing to 17 +/- 4 microM (TZR) and 37 +/- 6 microM (WT). A similar enhanced sensitivity to NU1025 (approximately 2.7-fold) was obtained when TZR cells were co-exposed to MNNG + NU1025. TM-induced DNA strand breaks were increased by co-incubation with NU1025, and again the TZR cell line showed increased sensitivity to NU1025. There were no significant changes in NMNAT activity in response to MNNG treatment over 24 h, either in the presence or in the absence of NU1025. These data demonstrate that modest decreases in cellular NAD levels can sensitize cells to alkylating agents and PADPRP inhibitors.


We have investigated the mechanism of resistance to adriamycin (ADR) of 3 human glioma cell lines in culture. The cell lines had different inherent sensitivities to ADR. Verapamil increased the ADR sensitivities of the 2 most resistant cell lines (G-UVW and G-CCM) by up to 5-fold. This effect was not seen in a sensitive cell line (G-MCF). Although the accumulation of ADR in the 3 cell lines was not related to inherent sensitivity, energy deprivation or the addition of verapamil produced an increase (up to 46%) in net uptake for both G-UVW and G-CCM, but not for G-MCF. For G-UVW the ADR efflux data were consistent with an energy-dependent ADR efflux mechanism which could be inhibited by verapamil. A similar mechanism was not found for G-CCM. In this cell line verapamil may act by increasing intracellular ADR binding. These data indicate that, while inherent resistance to ADR may be multifactorial, one possible mechanism of resistance in human glioma may involve changes in drug accumulation and/or binding as has been seen in animals models. A potential clinical role for verapamil in overcoming drug resistance in human solid tumours is also indicated.


A panel of human lung carcinoma lines representing the four main histological types , and derived from both primary and metastatic sites, has been established in xenograft and in tissue culture. The highest take rates were achieved when biopsy specimens were obtained from large tumour masses and cultured lines were most readily established after preliminary passages as xenografts. The established lines exhibited an overlapping spectrum of biochemical and morphological characteristics, and showed a tendency to change from one cell type to another, in keeping with the concept of a common endodermal cell of origin. Radiation resistance appeared to be related to the large-cell phenotype.


Eicosapentaenoic acid (EPA) has been shown to have an inhibitory effect on the growth of several pancreatic cancer cell lines in vitro. This study investigates the mechanism of growth inhibition and cytotoxicity of EPA on the pancreatic cancer cell line MIA PaCa-2. Cells were analysed for cell count, viability, cell cycle distribution and ultrastructural changes. There was a time- and dose-dependent decrease in cell count and viability in cultures of pancreatic cancer cells supplemented with EPA. Flow cytometric DNA analysis of MIA PaCa-2 cells incubated with EPA demonstrated the presence of sub G1 populations corresponding to the presence of apoptotic cells and the blockade of cell cycle progression in S-phase and G2/M-phase. The presence of apoptosis in EPA-supplemented cultures was further confirmed by DNA fragmentation and ultrastructural changes associated with apoptosis. Therefore, we conclude that EPA mediates its effect on the pancreatic cancer cell line MIA PaCa-2, at least in part, via cell cycle arrest and the induction of apoptosis.


Up regulation of genes which contribute to store operated channel (SOC) activity may plausibly be necessary for these processes to take place efficiently. TRPC proteins constitute a family of conserved Ca2+-permeable channels that have been shown to contribute to SOC activity.CaIn breast cancer biopsy tissues, TRPC3 and TRPC6 were the predominant TRPC genes expressed with TRPC3 and TRPC6 being significantly up regulated compared to normal breast tissue. In the lowly metastatic breast cancer cell line MCF-7, TRPC6 was the chief TRPC gene expressed while in the highly metastatic breast cancer cell line MDA-MB-231 both TRPC3 and TRPC6 were the predominant TRPC genes expressed. Western blotting, immunoconfocal analysis and immunoprecipitation experiments confirmed that the MDA-MB-231 cell line expressed both TRPC3 and TRPC6 protein with the majority of protein being intracellular. TRPC3 and TRPC6 were found to be in an immunoprecipitatble complex and co-localize within the cell. To demonstrate the potential of targeting TRP channels in breast cancer, hyperforin reportably a specific activator of TRPC6 significantly reduced the growth and viability of the breast cancer cell lines but had no effect on the non-cancerous breast cell line. Silencing of TRPC6 in MDA-MB-231 cells resulted in a significant reduction in cell growth but not viability.TRPC channels may be potential future targets for breast cancer diagnosis and therapy and deserve further investigation to evaluate their role in cancer cell physiology. Both are likely to have a significant role in the cancer process and Ca2+ is a regulator of proliferation and apoptosis  were added to the wells and incubated at 37°C for 2 h. The solution was removed and replaced by 890 μl of DMSO and 110 μl of Glycine buffer  for 10 mins. The numbers of viable cells were then quantified by measuring the absorbance at 570 nm with a spectrophotometer.P < 0.05.Data were analyzed by one-way ANOVA. Differences were taken as statistically significant when Our aims were to determine which TRP channel sub-types are expressed in normal and cancer breast biopsy tissues. mRNA was prepared from seven breast tissue samples obtained by biopsy of which three were found to be normal and four were diagnosed as cancerous. The mRNA was converted to cDNA and TRPC sub-type specific primers table  were utiBreast cell lines derived from cancer tissues are useful tools to understand the molecular factors involved in cancer and metastasis. In this study we chose three breast cell lines to investigate TRPC expression: MCF-10A (a non cancerous/transformed cell line), MCF-7 (lowly-metastatic cancer cell line) and MDA-MB-231 (highly-metastatic cancer cell line). mRNA was prepared from these cell lines and converted to cDNA. PCR amplification using TRPC sub-type specific primers was utilized and the PCR products were separated on agarose gels and visualized with a DNA stain Fig . PCR privisa versa. No TRPC3/TRPC6 complexes were observed in the absence of an immunoprecipitating antibody or with isotype control antibodies.Extracts of protein were made from MCF-10A, MCF-7 and MDA-MB-231 breast cell lines and separated by SDS-PAGE. Following blotting to nitrocellulose the blots were probed with antibodies to TRPC6 Fig  and TRPCOur PCR and protein results suggested that TRPC6 and/or TRPC3 were the predominant TRPC sub-types expressed as mRNA in the MCF-7 and MDA-MB-231 cell lines. To identify the localization of these TRPC channels in these cell lines we utilized immunochemistry staining with TRPC6 and TRPC3 specific antibodies. The MCF-7 and MDA-MB-231 cells were fixed and subsequently stained with a concanavalin-FITC conjugate. Following permeabilization the cells were probed with antibodies specific to TRPC6 or TRPC3 and visualized by confocal microscopy Fig , n = 4. Hyperforin was reported as a specific activator of TRPC6 compared to other TRPC channels . Since T2+ signalling to regulate pathways such as proliferation and apoptosis suggests that therapies that modulate Ca2+ signalling in cancer cells might be a therapeutic option [2+ channels and pumps that are aberrantly expressed in cancer cells might sufficiently disrupt Ca2+ homeostasis to selectively target cancer cells [2+channels are expressed in cancer cells? To date there is incomplete information as to the expression of TRP channels in normal and cancer breast tissues. Our major conclusions from this research are listed below:The ability of Cac option . Modulater cells . The pri1. Our experiments suggested that normal breast tissues expressed the TRPC1 and TRPC3 genes. Comparison of normal and breast cancer biopsy tissues indicated a significant alteration in TRPC channel expression both in terms of channel type expressed and levels of channel expression. In breast cancer biopsy tissues TRPC6 was expressed and appeared to be the predominant TRPC channel expressed. Additionally TRPC3 appeared to be significantly up regulated in breast cancer tissues. Of course our experiments could not distinguish which cell types these TRPC channels were expressed in, since the breast tissue could contain a variety of cell types.2. In the epithelial non-cancerous cell line MCF-10A only relatively very low amounts of TRPC1 expression was observed. In lowly metastatic breast cancer cell line MCF-7 the predominant channel expressed was a short splice variant of TRPC6. In the highly metastatic breast cancer cell line MDA-MB-231 the predominant TRPC channels expressed were TRPC3 and TRPC6 (of longer splice form variants). The amounts of TRPC3 and TRPC6 expressed in the MDA-MB-231 cell line were comparable in level, which leads us to speculate that they may form heteromultimers. The sizes of PCR products for TRPC3 and C6 found in the cancer tissues appeared to be consistent to that found in the MDA-MB-231 cell line.3. Protein expression of TRPC3 and TRPC6 in breast cancer cell lines appeared to be consistent with the PCR results. TRPC6 and TRPC3 expression was observed in the MDA-MB-231 cell line with the majority of protein being expressed in an intracellular location.4. Our data provides strong evidence that TRPC3 and TRPC6 form heteromutimeric channels in breast cancer epithelial cell line MDA-MB-231, which is consistent with previous reports of TRPC3/6 expression .2+ signalling hence affecting cell proliferation. In an earlier study, hyperforin induced apoptosis and inhibited the growth of various human and rat tumour cell lines in vivo, [5. Hyperforin a specific activator of TRPC6 produced a significant reduction in cell growth for the breast cancer cell lines, while the non-cancerous breast cell line was unaffected. Viability was significantly reduced in the breast cancer cell lines. TRPC6 activation is known to promote cell proliferation. This is an opposite result which is obtained here when using hyperforin however it may be expected that overactivation of TRPC6 by hyperforin could cause a disruption in Cain vivo, . Our res6. Complete silencing of TRPC6 in MDA-MB-231 cells using transfected SiRNA resulted in a significant reduction in cell growth but not viability.et al [et al [2+ current described in these cells. We propose that TRPC6 is the major TRPC channel expressed in MCF-7 cells. However correlation of the permeation and pharmacological profiles of the cationic current in MCF-7 cells as described by El Hiani et al, [et al [2+-permeable cation channels which are activated in response to agonist-stimulated PIP(2) hydrolysis and subsequent studies have provided substantial evidence that some TRPCs contribute to SOC activity [2+ is known to be involved in a number of metastatic processes including motility and proliferation [On the whole our results with the MCF-7 cell line confer with the recent report by Bolanz et al  who founl [et al  who suggi et al,  with tho, [et al  demonstrferation  these prThe authors declare that they have no competing interests.EA contributed to the siRNA and growth curves and helped to draft the manuscript. MD participated in its design and coordination. SK performed the PCR and immunoflurescence. CPP performed the remainder of assays conceived and participated in its design and coordination and drafted the manuscript.


P< 0.05), although to varying degrees, suramin and Å5 almost completely and N-acetylcysteine the least. In conclusion, this model shows the urokinase system is important for bladder invasion and can be used to investigate other mechanisms of bladder cancer invasion and also for the testing of intravesical drugs. © 2001 Cancer Research Campaign http://www.bjcancer.comAs well as being a passive support, the extracellular matrix also regulates key biological processes such as invasion, differentiation and angiogenesis. We have therefore developed an in vitro model of bladder cancer invasion using de-epithelialized rat bladder to allow for tumour cell–extracellular matrix interactions. Onto this we have seeded a panel of human bladder cancer cell lines ) representing progression from well to poorly differentiated phenotypes and used as models of superficial to invasive bladder cancer. The better differentiated cell lines RT4 and RT112 reproducibly grew as stratified epithelium, whereas poorly differentiated EJ28 cells invaded across a broad front. Invasion was not simply related to proliferation rate, measured either as doubling time on plastic (non-invasive 253J and invasive EJ28 having the same doubling time) or by Ki-67 proliferation index within the model. We used the model to test the ability of 4 compounds that interfere with tumour cell–extracellular matrix interactions  to inhibit invasion. At non-toxic concentrations, all significantly inhibited invasion (


Cell lines constitute a powerful model to study cancer, and here we describe three new epithelial ovarian cancer (EOC) cell lines derived from poorly differentiated serous solid tumors , as well as the matched ascites for one case (OV-1946).In addition to growth parameters, the cell lines were characterized for anchorage independent growth, migration and invasion potential, ability to form spheroids and xenografts in SCID mice.TP53 mutations were found in all cell lines, with TOV-1946 and OV-1946 harboring the same mutation, and none harbored the commonly observed somatic mutations in BRAF, KRAS or germline BRCA1/2 mutations found to recur in the French Canadian population. Conventional cytogenetics and spectral karyotype (SKY) analyses revealed complex karyotypes often observed in ovarian disease.While all cell lines were capable of anchorage independent growth, only the TOV-1946 and OV-1946 cell lines were able to form spheroid and produce tumors. Profiling of keratins, p53 and Her2 protein expression was assessed by immunohistochemistry and western blot analyses. Somatic This is the first report of the establishment of matched EOC cell lines derived from both solid tumor and ascites of the same patient. Epithelial ovarian cancer (EOC) is often described as the silent killer or the disease that whispers mainly due to absence of symptoms. This combined with the lack of specific/sensitive markers and/or techniques of screening leads to the diagnosis at late stages of the disease in more than 70% of patients. Unfortunately, the five year survival rate at this point of the disease is less than 30% . AlthougEOC is a complex disease stratified according to histopathological and morphological criteria. The majority of EOCs are thought to arise from the ovarian surface epithelium (OSE) that is derived from the coelomic epithelium. OSE is composed of multipotent cells that can differentiate and give rise to tumors of different histopathology types ,2. The lOver the past years several laboratories, including ours , have esIn this study, we describe three new serous EOC cell lines that were derived in our laboratory from either solid tumors or ascites of two chemotherapy-naïve patients. This is the first report characterizing cell lines derived from both solid tumor and ascites of the same patient. Moreover, the molecular and growth characteristics of the three cell lines present some unique features thereby providing the research community with new tools in the study of different aspects of serous EOC.Tumor samples were collected and banked following surgeries performed within the Division of Gynecologic Oncology at the Centre hospitalier de l'Université de Montréal . The study was approved by the CHUM institutional ethics committee and written consent was obtained from patients prior to sample collection. Stage was determined at the time of surgery. Histopathology and tumor grade were assigned by a pathologist according to the International Federation of Gynecology and Obstetrics (FIGO) criteria .2 and 5% O2.All primary cultures and cell lines were cultured in OSE medium  supplemented with 10% fetal bovine serum (FBS), 2.5 μg/ml amphotericin B and 50 μg/ml gentamicin. Cells were incubated in 5% COThe TOV-1946 cell line was established using the previously described scrape method on tumor tissue from patient 1946 ,43. BrieThe OV-1946 cell line was established from a mass of cells from the ascites of patient 1946. The mass was macro-dissected into small pieces, which were kept in a 100 mm petri dish for 27 days at which point adherent cells reached 80% confluence. Pieces of tissue were then discarded. Cells were divided in a proportion of 2:3 every week for the first 15 passages and then 1:2 twice a week until passage 70. Cells were then maintained and divided in a proportion of 1:5 twice a week.The TOV-2223 cell line was established from patient 2223 tumor tissue using the collagenase method. Briefly, tumor tissue was macro-dissected onto a 100 mm petri dish containing serum free OSE medium supplemented with 1000 U of collagenase . After 3–4 hours at 37°C, cells were resuspended into 8 ml of medium and the remaining tumor tissue pieces were discarded. The medium containing cells was then divided into four 60 mm petri dishes and 10% FBS was added. Cells were divided 1:2 once a week until passage 19 and then twice a week until passage 70. Subsequently, cells were maintained and divided in a proportion of 1:3 twice a week.For immunohistochemistry and western blot analyses, the following antibodies were used: beta actin AC-15 , p53 (D0-1) , anti-c-ErbB2/c-Neu , Keratin 19 Ab-1 , Keratin 7 Ab-2 (MS-1352-P0) and Keratin 8 Ab-4 .2O2 treatment was used to eliminate endogenous peroxidase activity. The sections were blocked with a protein blocking serum-free reagent  and incubated with different antibodies for 60 min at room temperature.Formalin fixed paraffin embedded tumors were sectioned at 4 μm and the slides were stained using the immunoperoxidase method. Briefly, tissue sections were heated at 60°C for 30 minutes, deparaffinized in toluene and rehydrated in an ethanol gradient. Slides were submerged in boiling citrate buffer (0.01 M citric acid adjusted to pH 6.0) and microwaved for 10 min to unmask antigens. A 3% H2O2 as a peroxidase substrate. Nuclei were counterstained with hematoxylin and all sections were observed by light microscopy at 400× magnification. Substitution of the primary antibody with phosphate buffered saline served as a negative control.The optimal concentration for each primary antibody was determined by serial dilutions. Tissues were incubated with either a secondary biotinylated antibody  or a rabbit anti-goat biotin-conjugated antibody (1:300)  for 20 min followed by incubation with a streptavidin-peroxidase complex  for 20 min at room temperature. Reaction products were developed using diaminobenzidine containing 0.3% H5 cells were seeded onto 60 mm petri dishes. On day 1, 3, 5, 7, 9, 11 and 13 the cells were trypsinized, resuspended in medium and counted using a hemacytometer. Each experiment was performed in triplicate for each harvest and repeated once. Saturation density was defined as the mean maximum number of cells at confluence counted from two independent experiments performed with triplicates and the doubling time was calculated according to the slope of the linear portion of the growth curve.Growth rates were assessed as previously described . On day 4 cells in agarose (0.33 g/100 ml OSE complete medium for the upper layer and 0.66 g/100 ml OSE complete medium for the base layer) dATP ; 1× PCR buffer ; 2.5 nmol each dCTP, dGTP and dTTP; 0.3 nmol dATP; 1.5 mM MgCl2; 15 pmol of each primer [h primer ; and 0.5BRCA1 and 8765delAG, 6085G>T and 3398delAAAAG in BRCA2 were investigated in DNA from patient matched peripheral blood lymphocytes as described [The common French Canadian founder mutations 4446C>T and 2953delGTAinsC in escribed .® system (ASI) mounted on a Zeiss Axioplan II microscope and analyzed using the SkyView version 1.6.1 software (ASI).Metaphase preparation and cytogenetic analyses with a trypsin-Giemsa banding technique of the TOV-2223, TOV-1946 and OV-1946 ovarian cancer cell lines were performed according to standard cytogenetic procedures. Clonal chromosomal abnormalities and GTG-banded karyotypes were described according to the International System for Human Cytogenetic Nomenclature . MetaphaCell lines were derived from both solid tumors (TOV-1946 and TOV-2223G) and ascites (OV-1946) of two chemotherapy naive patients. Patients 1946 and 2223 from which the cell lines were derived both presented poorly differentiated (grade 3) serous papillary cystadenocarcinoma at stage IIIC. Based on residual tumor present following surgery, both patients were considered to be suboptimaly debulked. Patient 2223 received a palliative treatment only and survived for 18 months while patient 1946 died from post-operative complications. Patient 1946 did not have any known familial history of cancer. However, two sisters of patient 2223 were diagnosed with ovarian cancer (70 and 80 years old) and numerous colon cancers were diagnosed within the family. Clinical data for both patients are summarized in Table At initial passage all three cell lines appeared more (TOV-1946 and TOV-2223) or less (OV-1946) heterogenous with populations of cells with obvious fibroblastic contaminants Figure . With suHematoxylin-Eosin stained tumor tissue sections of patient 1946 and 2223 Figure . This reOne of the hallmarks of cancer cells is their ability to grow in the absence of exogenous growth factors, which can be verified by culturing cancer cells in low serum conditions. All cell lines were able to grow in a medium containing only 1% of FBS. In general however, growth rates were slower in low serum conditions than those observed in a typical 10% FBS environments (data not shown).in vivo in the original tumor  analysis was limited to the recurrent mutations found in women of French-Canadian descent as cell lines were derived from patients of this ancestral origin [TP53 were identified in the DNA from all three EOC cell lines  [TP53 mutation analysis revealed no evidence of heterozygosity, it is likely that no functional p53 is encoded in these cell lines. No mutations in the other genes studied were found in the cell lines.In order to further characterize the cell lines, we performed somatic mutation analysis of database ,58. The s Figure  and 2I. atabase) . As TP53The cytogenetic alterations of the three cell lines were assessed by G-banded karyotyping and by spectral karyotyping (SKY). Twenty-five metaphases in G-banding and thirty-two metaphases in SKY (TOV-2223), twenty-two metaphases in G-banding and thirty-nine metaphases in SKY TOV-1946) and nineteen metaphases in G-banding and fifteen metaphases in SKY (OV-1946) were analyzed. The G-banded analysis of the three cell lines revealed a modal number of 51 to 71 chromosomes with complex karyotypes that are described as a composite karyotype containing the clonal chromosomal abnormalities . The homogeneity of cells observed at passage 0 of OV-1946 could reflect the fact that these cells came from a mass in the patient's ascites that is more homogenous then a tumor mass often composed of, among others, epithelial tumor cells, stromal cells and endothelial cells.Cell lines were also evaluated on their capacity to survive and grow in low serum conditions. It is worth noticing that although in normal growth conditions both TOV and OV-1946 cell lines present similar growth rates, in low serum conditions the later is almost two times slower. This could be in line with the fact that the OV-1946 cell line comes from ascites which is rich in growth factors and in culture can replace FBS . This ceKRT7, KRT8 and KRT18. In order to determine if our cell lines presented these EOC markers, we monitored protein expression of Krt7, Krt8 and Krt18 by western blot. When antibodies were appropriate for immunohistochemistry on paraffin embedded tissues, we also compared keratins expression in the original tumor tissues. It is also well described that monitoring expression of Krt7 and Krt20 could distinguish between ovarian and gastro-intestinal tract tumors . Both tHER2 gene leading to the overexpression of the protein Her2 . Theiewed in -67. In Eiewed in -63). AllDifferences in growth rates, migration, invasion and spheroid formation between the TOV-1946 and TOV-2223 underline the diversity of phenotypes that can be observed within the same type of serous EOC disease. The results from the comparison of the TOV-1946 and OV-1946 growth characteristics, demonstrate that these cell lines present with unique phenotypes. Indeed, TOV-1946 cells had a better capability to invade and migrate although their capacity to form spheroid is reduced compared to the OV-1946 cells. One might speculate that cells derived from solid tumor conserve their migration and invasion property but in ascites, these characteristics are less vital. It has been previously shown that cells present in ascites form spheres or aggregates that can adhere to different extracellular matrices as well as to normal human mesothelial cells  but are in vivo tumorigenicity can usually be predicted by the ability of the cells to grow in soft agar [in vivo [As both the TOV-1946 and OV-1946 injected mice developed ascites we hypothesize that the ability for cell lines to induce ascites formation in mice is intrinsic to the given cell line and independent of their origin (solid tumor versus ascites). It has been previously shown that the oft agar . The OV-[in vivo . ConsistThe unique features of the described cell lines provide complementary models for different aspects of the disease. For example, while TOV-2223 and the TOV-1946 cell lines are both derived from solid tumors of the same type of serous EOC disease, they vary in important aspects including their aggressiveness and karyotype. They may prove useful for comparative studies to uncover molecular events that distinguish very aggressive from more indolent serous disease in ovarian cancer. Although the TOV-1946 and OV-1946 lines were derived from the same patient, they were collected from the solid tumor and the corresponding ascites respectively. Although common genome modification are shared between these two cell lines  , the wound-healing assays, mice assays as well as writing and editing of the manuscript according to all authors revisions. LP derived and established the new cell lines from tumor samples and performed the immunohistochemistry on the primary cultures and cell lines. JM performed the immunohistochemistry on tumor tissue. JL performed the Western blot.M–LP performed the invasion assays. SLA and ZS performed mutations analyses. JH analyzed the SKY and G-banding assays. PNT, DMP and A–MM–M contributed to the conception and design of the study as well as analysis and interpretation of the data. All authors revised the manuscript and gave final verbal approval.The pre-publication history for this paper can be accessed here:A) G-banded metaphases from the TOV-2223 cell line (cells 15 and 36 respectively). Arrows indicate the abnormal chromosomes, mar: marker chromosome. B) Combined inverted-DAPI and SKY image of cell 45 and 46 respectively from the TOV 2223 cell line with identification of some marker chromosomes.Click here for fileA) G-banded metaphases from the TOV-2223 cell line (cells 15 and 36 respectively). Arrows indicate the abnormal chromosomes, mar: marker chromosome. B) Combined inverted-DAPI and SKY image of cell 45 and 46 respectively from the TOV 2223 cell line with identification of some marker chromosomes.Click here for fileA) G-banded metaphases from the TOV-1946 cell line (cells 6 and 43 respectively). Arrows indicate the abnormal chromosomes, mar: marker chromosome. B) Combined inverted-DAPI and SKY image of cell 44 and 36 respectively from the TOV-1946 cell line with identification of some marker chromosomes.Click here for fileA) G-banded metaphases from the TOV-1946 cell line (cells 6 and 43 respectively). Arrows indicate the abnormal chromosomes, mar: marker chromosome. B) Combined inverted-DAPI and SKY image of cell 44 and 36 respectively from the TOV-1946 cell line with identification of some marker chromosomes.Click here for fileA) G-banded metaphase from the OV-1946 cell line (cell 24). Arrows indicate the abnormal chromosomes, mar: marker chromosome. B) Combined inverted-DAPI and SKY image of cell 11 from the OV-1946 cell line with identification of some marker chromosomes.Click here for file


The extensive chromosome rearrangements of breast carcinomas must contribute to tumour development, but have been largely intractable to classical cytogenetic banding. We report here the analysis by 24-colour karyotyping and comparative genomic hybridization (CGH) of 19 breast carcinoma cell lines and one normal breast epithelial cell line, which provide model examples of karyotype patterns and translocations present in breast carcinomas. The CGH was compared with CGH of 106 primary breast cancers. The lines varied from perfectly diploid to highly aneuploid. Translocations were very varied and over 98% were unbalanced. The most frequent in the carcinomas were 8;11 in five lines; and 8;17, 1;4 and 1;10 in four lines. The most frequently involved chromosome was 8. Several lines showed complex multiply-translocated chromosomes. The very aneuploid karyotypes appeared to fall into two groups that evolved by different routes: one that steadily lost chromosomes and at one point doubled their entire karyotype; and another that steadily gained chromosomes, together with abnormalities. All karyotypes fell within the range seen in fresh material and CGH confirmed that the lines were broadly representative of fresh tumours. The karyotypes provide a resource for the cataloguing and analysis of translocations in these tumours, accessible at http://www.path.cam.ac.uk/~pawefish. © 2000 Cancer Research Campaign


The most cytotoxic species is Cp(CpCO-NH-C6H4-(CH2)2CH3)TiCl2 with an IC50 of 24(2) μM, followed by Cp(CpCO-NH-C6H4-Br)TiCl2, IC50 of 46(4) μM and Cp(CpCO-NH-C6H4-OCF3)TiCl2, IC50 of 49(6) μM. There is no correlation between the nature of the para substituent on the phenyl ring and the cytotoxic properties on MCF-7 cell line.A new titanocenyl amide containing flavone as pendant group has been synthesized by reaction of titanocenyl carboxylic acid chloride and 7-Aminoflavone and structurally characterized by spectroscopic methods. This species and eight previously synthesized titanocenyl amide complexes have been tested in breast adenocarcinoma cancer cell line, MCF-7. The functionalization of titanocene dichloride with amides enhances the cytotoxic activity in MCF-7. Two sets of titanocenyl amides can be identified, with IC The fact that it possesses antitumor properties in cancer cell lines that are insensitive to cisplatin as well as lower toxic effects than cisplatin, has motivated the scientific community to continue investigating this species 2TiCl2 · 2HCl, on MCF-7 with an IC50 of 62 μM . Other types of functionalized titanocenes have been reported with enhanced cytotoxic properties and ICar range –22. For of 62 μM . Our mor7), with a propyl group on the para position of the phenyl ring, exhibited the highest cytotoxic activity. Finally, other strategies are currently being pursued such as replacement of chlorides by fluorides as ancillary ligands on Titanocene Y and derivatives [Beckhove and co-workers have reported anticancer properties of bis-[p-methoxybenzyl)cyclopentadienyl] titanium dichloride (Titanocene Y). This is active against a wide variety of cancer cell lines. Moreover, Titanocene Y has been tested in explanted human breast tumor cells and in xenografted MCF-7 tumors in mice with promising results in terms of doses, low toxicity, and reduction of tumor volume . Also itivatives . It has ivatives .The functionalization of titanocene dichloride with amides (phenyl amides) increases the cytotoxic activity of the resulting titanocenyl amide complexes. One possible explanation could be the Ti-O(amide) bond which provides stability in aqueous environment and makes the complex more resistant to hydrolysis. Although this is highly speculative, based on the structural features of these titanocenyl amides and the hypothesis that albumin is the carrier protein of titanocene into the target place inside the cell , 25, we Comparative Table of Titanocenyl Amides Complexes Between Colon Cancer HT-29 and Breast Cancer MCF-7 Cell Lines.Click here for additional data file.


The single-crystal X-ray structure of complex [SnPh2(L)(DMSO)] (5) shows that the ligand is doubly deprotonated and is coordinated as tridentate ligand.The six coordination number is completed by two carbon atoms of phenyl groups. There are two similar monomers 5a (Sn1) and 5b (Sn51) in the asymmetric unit. The monomers 5a and 5b are linked through intermolecular hydrogen bonds of N–H–O and C–H–S type. C–H → π, intermolecular interactions, intra- and intermolecular hydrogen bonds stabilize this structure and leads to aggregation and asupramolecular assembly. The IR and NMR  spectroscopic data of the complexes are reported. The in vitro cytotoxic activity has been evaluated against the cells of three human cancer cell lines: MCF-7(human breast cancer cell line), T-24 (bladder cancer cell line), A-549  and a mouse L-929 (a fibroblast-like cell line cloned from strain L). Compounds 1, 3, and 4 were found active against all four cell lines. Selectivity was observed for complexes 3 and 4 which were found especially active against MCF-7 and T-24 cancer cell lines.The synthesis and spectral characterization of novel diorganotin complexes with 3-hydroxypyridine-2-carbaldehyde thiosemicarbazone, H Organotin(IV) compounds find wide applications as catalysts and stabilizers, and certain derivatives are used as biocides, as antifouling agents and for wood preservation. It has been observed that several diorganotin adducts show potential as antineoplastic and antituberculosis agents –4. 1) is a member of the so-called α-(N)-heterocyclic carbaldehyde thiosemicarbazones (HCTs), which are the most potent known inhibitors of ribonucleoside diphosphate reductase. Compounds (1) and 5-hydroxypyridine-2-carbaldehyde thiosemicarbazone have shown high anticancer activity in animal models but were found to be readily glucuronidated and rapidly excreted  have been studied by us  (5). IR and NMR spectroscopic data are reported. The results of the cytotoxic activity of diorganotin complexes have been evaluated for antiproliferative activity in vitro against human cancer cell lines: MCF-7, T-24, A-549, and a mouse L-929.The present paper includes the interaction of SnPh−1) and nujol mulls dispersed between polyethylene disks (400–40 cm−1). The free ligand was dissolved in (CD3)2SO and 1H, 1H–1H COSY and 13C spectra were acquired on a BRUKER 300 MHz spectrometer. Compounds 2–4 were dissolved in CDCl3 and were spectroscopically characterized by the use of 1D and 2D NMR spectroscopy on a Varian 600 MHz spectrometer. Experimental data were processed using VNMR and WinNMR routines. Chemical shifts (δ) are reported in ppm while spectra were referenced by the standard experimental setup. 119Sn NMR spectra were acquired on the Varian 600 MHz and tin spectra were referenced by the use of external solution of Me4Sn in C6D6. The splitting of proton resonances in the reported 1H-NMR spectra is defined as s = singlet, d = doublet, t = triplet, and m = multiplet. The chemical shifts are reported in ppm for 1H and 13C NMR. Elemental analyses were carried out by the microanalytical service of the University of Ioannina, Greece.All reagents were commercially available (Aldrich or Merck) and used as supplied. Solvents were purified and dried according to standard procedures. Melting points (m.p.) were determined in open capillaries and are uncorrected. IR and far-IR spectra were recorded on a Perkin Elmer Spectrum GX Fourier transform spectrophotometer using KBr pellets (4000–400 cm−1): 3555 s, 3451 m v(OH); 3291 m, 3194 m, v; 1639 s, v(C=N); 1229 s, v(C–O); 1098 s, v(NN); 827 s, v(C=S). 1H-NMR: 11.50 ; 9.62 ; 8.12 , 3J = 4.2 Hz); 7.26  3J = 8.5, 4J = 4.2 Hz); 7.30  3J = 8.4, 4J = 1.4 Hz); 8.34 ); 8.00, 8.24 . 13C-NMR: 178.0 C(7); 153.1 C(4); 144.4 C(6); 141.1 C(1); 137.6 C(5); 125.3 C(2); 124.2 C(3). Anal. calc. for C7H8N4OS (196.04): C, 42.8; H, 4.1; N, 28.6; S, 16.3; found: C, 42.6; H, 3.9; N, 28.2; S, 16.0%.The ligand was synthesized according to a published procedure . The whi−1): 3296 m, v(NH2); 1580 s, v(C=N); 1176 s, v(C–O); 1111 s, v(NN); 804 w, v(C=S); 583 m, 564 v(Sn–C); 431 m, v(Sn–N); 231 m, v(Sn–O); 376s, ν(Sn–S). 1H-NMR: 8.09 , 3J = 4.2, 4J = 1.4 Hz); 7.18  3J = 8.5, 4J = 4.2 Hz); 7.08  3J = 8.5, 4J = 1.4 Hz); 8.76 ); 5.09 ; 0.89  = 35.7 Hz). 13C-NMR: 168.8 C(7); 163.4 C(4); 161.5 C(6); 140.2 C(1); 135.4 C(5); 127.6 C(2); 128.9 C(3); 6.22  = 309 Hz). 119Sn NMR: δ = −94.5. Anal. calc. for C9H12N4OSSn (343.0): C, 31.5; H, 3.5; N, 16.3; S, 9.3; found: C, 31.5; H, 3.7; N, 16.0;, S, 9.1 %.Dimethyltin(IV) oxide  and 3-hydroxypyridine-2-carbaldehyde Thiosemicarbazone  in benzene (20 mL) were stirred and were refluxed for 12 hours under azeotropic removal of water (Dean-Stark trap). The resulting clear solution was rotary evaporated under vacuum to a small volume (2 mL), chilled and triturated with diethyl ether to give a white solid. The powder was recrystallized from distilled diethyl ether and dried in vacuo over silica gel to give yellow solid; mp. 228–230°C, Yield 35%. IR (cm−1): 3292 m, v(NH2); 1577 s, v(C=N); 1173 s, ν(C–O); 1114 s, v(NN); 805 w, ν(C=S); 578 ms, 560 sh ν(Sn–C); 418 m, ν(Sn–N); 247 m, ν(Sn–O); 394 ms, ν(Sn–S). 1H-NMR: 8.05 , 3J = 4.2, 4J = 1.3 Hz); 7.16  3J = 8.5, 4J = 4.2 Hz); 7.07  3J = 8.5, 4J = 1.3 Hz); 8.82 ); 5.22 ; 0.87 ; 1.32 ; 1.54, 1.64 . 13C-NMR: 167.7 C(7); 162.5 C(4); 160.0 C(6); 138.7 C(1); 134.5 C(5); 126.8 C(2); 128.0 C(3); 26.9  = 265 Hz); 28.2 (Cβ); 27.3 (Cγ); 14.6 (Cδ). 119Sn NMR: δ = −194.4. Anal. calc. for C16H18N4OSSn (537.2): C, 42.2; H, 5.7; N, 13.1; S, 7.5; found: C, 42.0; H, 5.9; N, 13.2; S, 7.4 %.Dibutylltin(IV) oxide  and 3-hydroxypyridine-2-carbaldehyde Thiosemicarbazone  in benzene (20 mL) were stirred and were refluxed for 12 hours under azeotropic removal of water (Dean-Stark trap). The resulting clear solution was rotary evaporated under vacuum to a small volume (2 mL), chilled and triturated with diethyl ether to give a white solid. The powder was recrystallized from distilled diethyl ether and dried in vacuo over silica gel to give yellow solid; mp. 126–128°C, Yield 41%. IR (cm−1): 3269 m, ν(NH2); 1589 s, ν(C=N); 1185 s, v(C–O); 1118 s, v(NN); 808 m, v(C=S); 322 ms, 303 sh ν(Sn–C); 419 m, ν(Sn–N); 248 m, ν(Sn–O); 370 ms, ν(Sn–S). 1H-NMR: 8.02, 3J = 4.2 Hz); 7.18, 3J = 8.5, 4J = 4.2 Hz); 7.29 , 3J = 8.5 Hz); 8.88 ); 5.37 ; 7.82 ; 7.34–7.30 . 13C-NMR: 167.4 C(7); 164.0 C(4); 161.5 C(6); 140.2 C(1); 135.4 C(5); 127.8 C(2); 129.3 C(3); 142.1  = 424 Hz); 135.8  = 28 Hz); 128.8  = 37 Hz); 130.2  = 8.7 Hz); 119Sn NMR: δ = −227.2. Anal. calc. for C19H16N4OSSn (663.1): C, 48.9; H, 3.5; N, 12.0; S, 6.9; found: C, 48.6; H, 3.5; N, 10.7; S, 6.8%.Diphenylltin(IV) oxide  and 3-hydroxypyridine-2-carbaldehyde thiosemicarbazone  in benzene (20 mL) were stirred and were refluxed for 12 hours under azeotropic removal of water (Dean-Stark trap). The resulting clear solution was rotary evaporated under vacuum to a small volume (2 mL), chilled and triturated with diethyl ether to give a white solid. The powder was recrystallized from distilled diethyl ether and dried in vacuo over silica gel to give yellow solid : mp. 186–188°C, Yield 34%. IR  Sp. z o.o  programs. Crystal structure was solved by direct methods (program SHELXS97) and refined by the full-matrix least-squares method on all F2 data using the SHELXL97  and [Pd(Ac4Et)2] complexes can be characterized by cellular pharmacological properties distinctly different from those of cis-platin [2(Fo4Npypipe)] and [ZnCl2(Ac4Npypipe)] where Fo4Npypipe and Ac4Npypipe are the monoion of 2-formyl pyridine N(4)-1-(2-pyridyl)-piperazinyl thiosemicarbazone and 2-acetyl pyridine N(4)-1-(2-pyridyl)-piperazinyl thiosemicarbazone have been evaluated in vitro against MCF-7, T-24, A-549, and L-929 cell lines and it was found to exhibit remarkable antiproliferative activity with mean IC50 values of 0.2–20 μM [Complexes of s-platin . The com.2–20 μM . The dip.2–20 μM .cis-platin and diorganotin(IV) oxides. Results showed that the ligand as well as the complexes 3 and 4 demonstrated excellent antiproliferative activity, IC50 values range from 0.02–2.5 μM, against all cell lines tested, while for cis-platin the IC50 values range from 0.7–41 μM. The diorganotin(IV) oxides are nontoxic against L-929 and T-24 cancer cell lines and exhibit poor cytotoxic activity in A-549 and excellent antiproliferative activity against MCF-7 cancer cell line.The antiproliferative activity of compounds is presented in 1 is more cytotoxic compared to cis-platin against the A-549, T-24 and MCF-7 cell lines and less cytotoxic against L-929 cell lines. The diorganotin complex 2 is nontoxic against T-24 cell line, less cytotoxic against A-549 and L-929, and more cytotoxic against MCF-7 cell line compared to cis-platin. The diorganotin complexes 3 and 4 are in the same μM range compared to cis-platin against L-929 and A-549 cancer cell lines and more cytotoxic against T-24 and MCF-7 cancer cell lines. The IC50 values for the ligand, 1, against MCF-7 and T-24 cell lines are 0.15 μM and 2.09 μM, respectively, and against A-549 and L-929 cell lines are 0.81 μM and 2.5 μM, respectively. Ligand is 19.8 ± 1.3 and 52.4 ± 4.6 times more active than cis-platin against T-24 and MCF-7 cell lines, respectively. The IC50 values for 3 against MCF-7 and T-24 cell lines are 1.97 × 10−2 μM and 1.1 μM, respectively, and against A-549 and L-929 cell lines are 0.77 μM and 1.05 μM, respectively. Complex 3 is 403.6 ± 20.2 times more active than cis-platin against MCF-7 cell line and 37.9 ± 3.1 times more active than cis-platin against T-24 cell line. The IC50 values for 4 against MCF-7 and T-24 cell lines are 7.28 × 10−2 μM and 0.73 μM, respectively, and against A-549 and L-929 cell lines are 0.83 μM and 1.37 μM, respectively. Complex 4 is 110.3 ± 9.9 times more active than cis-platin against MCF-7 cell line and 57.1 ± 3.9 times more active than cis-platin against T-24 cell line. Compounds 1, 3, and 4 were found active against all four cell lines. Selectivity was observed for complexes 3 and 4 which were found especially active against MCF-7 and T-24 cancer cell lines. The mentioned evident differences in the antiproliferative action of the ligand and its diorganotin(VI) complexes indicate that these complexes really exist under the condition of the biological tests. Interestingly enough, 3 and 4 were found to be more potent cytotoxic agent than the prevalent benchmark metallodrug, cis-platin, under the same experimental conditions measured by us. The superior activity of 3 and 4 assumes significance in light of the fact that cis-platin is undisputedly the most studied and widely used metallopharmaceutical for cancer therapy known to date. It is noteworthy the high selectivity against MCF-7 and T-24 cancer cell lines.The ligand


A cDNA library generated from the β-TC3 SV40 T-antigen transformed insulinoma cell line was screened for members of this receptor family potentially relevant to both beta cell development and autoimmunity. Degenerate oligonucleotide primers to a consensus region of these receptors were used and the IL-11 receptor (α chain was identified. Despite confirmation of IL-11 receptor mRNA expression, iodinated bioactive IL-11 did not bind specifically to β-TC3 cells and gp-130-dependent cytokines did not elicit signalling events in beta cell lines. This was explained by absence of gp-130 protein or mRNA in the beta cell lines tested and in primary islets. We conclude from these resuits that the previously recognised effects of IL-6 family member cytokines on pancreatic islets must be indirect


A panel of 18 well characterised human small cell lung cancer (SCLC) cell lines was assessed for the production of adrenocorticotrophin (ACTH) and its precursor peptides, pro-opiomelanocortin (POMC) and pro-ACTH. These precursor peptides were measured directly using a novel two-site immunoradiometric assay (IRMA) based on monoclonal antibodies, in conjunction with a similar IRMA for ACTH 1-39. Significant concentrations of ACTH precursors were secreted by 10 of the 18 cell lines (56%). The low levels of ACTH immunoreactivity detected in seven cell lines could be accounted for by the known cross-reactivity of precursors in the ACTH IRMA. This suggests there is little, if any, processing of ACTH precursors to ACTH. Cell pellet extracts contained undetectable or low levels of ACTH precursors and ACTH, indicating that these peptides are not stored intracellularly. During the growth of the SCLC cells in vitro ACTH precursors accumulated progressively in the culture medium. Thus the combination of a direct assay for the ACTH precursors and the panel of SCLC cell lines provides a valuable in vitro model for the expression of POMC in human tumours.


The effects of single doses of ICRF 159 (Razoxane) on the cell-cycle kinetics of lines of BKH 21S cells in vitro were studied by means of flow micro-fluorometry (FMF). A characteristic accumulation of cells in the 4n DNA region after 6 h in the presence of ICRF 159 was evident in cells which were sensitive to the cytotoxic effects of the agents, as judged by colony-forming assays. The subsequent accumulation of 8n cells as the major population within 24 h indicates that this 6 h effect is associated with the induction of tetraploidy through abnormal mitosis, rather than reflecting a G2 block of potential value in combination therapy. In ICRF 159-resistant sublines, the cell-cycle distributions were similarly affected, but only the doses of the drug high enough to reduce their surviving fraction. Somatic cell hybrids from crosses between sensitive and resistant cells demonstrated intermediate responses in ICRF 159, both in terms of cell-cycle kinetics and impairment of reproductive integrity. These data suggest a relationship between the 2 manifestations of the cellular action of ICRF 159, and also a possible predictive role for FMF in the assessment of response to this particular agent.