Rhipicephalus (Boophilus) decoloratus, Rhipicephalus (Boophilus) microplus, Ixodes ricinus and Ixodes scapularis. For short-term cold storage, cells were kept under refrigeration at 6°C for 15, 30 and 45 days. For cryopreservation in liquid nitrogen, use of a sucrose-phosphate-glutamate freezing buffer (SPG) as cryoprotectant was compared with dimethylsulfoxide (DMSO) supplemented with sucrose. Cell viability was determined by the trypan blue exclusion test and cell morphology was evaluated in Giemsa-stained cytocentrifuge smears.Tick cell lines are now available from fifteen ixodid and argasid species of medical and veterinary importance. However, some tick cell lines can be difficult to cryopreserve, and improved protocols for short- and long-term low temperature storage will greatly enhance their use as tools in tick and tick-borne pathogen research. In the present study, different protocols were evaluated for cold storage and cryopreservation of tick cell lines derived from R. (B.) microplus, R. (B.) decoloratus, I. ricinus and I. scapularis cell lines; lines from the latter three species could be easily re-cultivated after 45 days under refrigeration. While cell lines from all four tick species cryopreserved with 6% DMSO were successfully resuscitated, the R. (B.) decoloratus cells did not survive freezing in SPG and of the other three species, only the R. (B.) microplus cells resumed growth during the observation period.Cold storage at 6°C for up to 30 days was successful in preserving R. (B.) decoloratus, R. (B.) microplus, and I. ricinus, and use of SPG as an alternative to DMSO for cryopreservation, thus making an important contribution to more reliable and convenient tick cell culture maintenance.This constitutes the first report on successful short-term refrigeration of cells derived from  Anaplasma, Ehrlichia and Rickettsia, and numerous arboviruses have been propagated in tick cell cultures  dimethylsulfoxide (DMSO), as described for the cryopreservation of IDE8 cells [ii) a sucrose-phosphate-glutamate freezing buffer (SPG) comprising 74.62 g/l sucrose, 0.517 g/l KH2PO4, 1.643 g/l K2HPO4.3 H2O and 0.907 g/l potassium glutamate [Four cell lines were used at cell densities ranging from 2.5 to 3.3 × 10E8 cells , and sup® Frosty™ Cryo 1°C freezing container, which resulted in a continuous decrease of temperature at a rate of one degree per minute. After being kept overnight at -80°C, the cryotubes were transferred into a liquid nitrogen tank, where they were stored in the liquid phase for at least seven days.For each cell line, cells harvested by pipetting from 12 to 15 tubes were pooled, and centrifuged at 400 × g for 10 minutes at 4°C. The cell pellet was resuspended in 5 ml of the respective medium and counted. Half of the cell suspension was held on ice, 2.5 ml of ice-cold medium containing 12% DMSO was added to give a final concentration of 6% DMSO and 1 ml aliquots were transferred to ice-cold 2 ml cryotubes. The other half of the cell suspension was centrifuged as before, the cell pellet was resuspended in 5 ml SPG and 1 ml aliquots were transferred to ice-cold 2 ml cryotubes. Thus three replicates were set up for each condition and each cell line. All cryotubes were frozen at -80°C using a NALGENEFor resuscitation, the cell suspensions were thawed rapidly by immersion of the cryotubes in a water bath at 37°C. Cells cryopreserved with DMSO were resuspended in 10 ml of the appropriate complete culture medium and were centrifuged at 400 × g at 4°C for 10 minutes. Pellets were resuspended in 1 ml of complete medium and transferred to culture tubes already preconditioned with 2 ml of the same medium for incubation. Cells cryopreserved in SPG were transferred immediately after thawing into culture tubes containing 2 ml of the appropriate complete culture medium. All tubes were incubated and maintained under the respective culture conditions for each cell line  decoloratus and both R. (B.) microplus cell lines by both protocols; therefore one cell line from each species was included in each protocol test.Refrigeration and cryopreservation protocols were tested on cell lines from each of the four tick species. Due to insufficient cell numbers at the start of the study, it was not possible to test both After storage under refrigeration, the BDE/CTVM14 cell line showed viability of over 80% in all replicates at all three storage periods , the only exception being on the first day after storage for 45 days. Viability rates remained almost constant during the entire experimental period and parent cultures and subcultures were nearly the same after all three storage periods Fig . MorpholThe BME/CTVM6 cell line showed good viability after storage at 6°C for 15 days, nearly as high as on day A Fig , whereasI. ricinus cell line IRE/CTVM20 appeared to be unaffected by a refrigeration period of 15 days, as viability over the 45-day period following re-cultivation was similar to that observed on day A  decoloratus and R. (B.) microplus can be successfully stored under refrigeration for at least 30 days. Furthermore, as an alternative to using DMSO as a cryoprotectant, R. (B.) microplus cells can be cryopreserved in a sucrose-phosphate-glutamate freezing buffer which allows culture propagation after resuscitation. These protocols will help to extend the use of tick cell lines as tools for tick-borne pathogen research by improving the ease and reliability of their storage at low temperatures.The results presented here show that cell lines derived from the ixodid tick species The authors declare that they have no competing interests.Rhipicephalus spp and Ixodes ricinus cell lines and critically revised the manuscript. EZ was involved in critically revising the manuscript for intellectual content. LP developed the conception and design of the study and was involved in drafting the manuscript. All authors have given final approval of the version to be publishedGL conducted all laboratory work and contributed to drafting the manuscript. LB-S provided the


Ribavirin (RBV) is a synthetic nucleoside analog with broad spectrum antiviral activity. Although RBV is approved for the treatment of hepatitis C virus, respiratory syncytial virus, and Lassa fever virus infections, its mechanism of action and therapeutic efficacy remains highly controversial. Recent reports show that the development of cell-based resistance after continuous RBV treatment via decreased RBV uptake can greatly limit its efficacy. Here, we examined whether certain cell types are naturally resistant to RBV even without prior drug exposure. Seven different cell lines from various host species were compared for RBV antiviral activity against two nonsegmented negative-strand RNA viruses, vesicular stomatitis virus  and Sendai virus . Our results show striking differences between cell types in their response to RBV, ranging from virtually no antiviral effect to very effective inhibition of viral replication. Despite differences in viral replication kinetics for VSV and SeV in the seven cell lines, the observed pattern of RBV resistance was very similar for both viruses, suggesting that cellular rather than viral determinants play a major role in this resistance. While none of the tested cell lines was defective in RBV uptake, dramatic variations were observed in the long-term accumulation of RBV in different cell types, and it correlated with the antiviral efficacy of RBV. While addition of guanosine neutralized RBV only in cells already highly resistant to RBV, actinomycin D almost completely reversed the RBV effect (but not uptake) in all cell lines. Together, our data suggest that RBV may inhibit the same virus via different mechanisms in different cell types depending on the intracellular RBV metabolism. Our results strongly point out the importance of using multiple cell lines of different origin when antiviral efficacy and potency are examined for new as well as established drugs in vitro. The mechanism of non-response to RBV/IFN treatment is very controversial and, unfortunately, no alternative therapies available for non-responders.de novo synthesis of GTP; ii) direct interaction of phosphorylated RBV with and inhibition of viral RNA polymerase, iii) RNA chain termination as a result of incorporation of RTP  into replicating RNA strands by viral RNA polymerases; iv) “error catastrophe” as a result of RTP incorporation into the viral genome paired with cytidine and uridine as a substitute for guanine and/or adenine, resulting in so called “lethal mutagenesis”, a meltdown of genetic information; v) inhibition of mRNA capping; and vi) immunomodulation of antiviral cellular responses such as the ability to induce a Th2 to Th1 shift in the immune response.The understanding of RBV treatment failures is complicated by an unclear mechanism of RBV action, partly due to its apparent pleiotropic nature Previous studies in search of explanations for RBV treatment failures were largely focused on the role of viral determinants of RBV resistance While drug resistant viral mutants may explain at least some failures with RBV treatments, recent reports propose that cell-based resistance to RBV could be an important factor explaining the low antiviral activity of RBV in at least some experimental and clinical systems Mononegavirales), vesicular stomatitis virus  and Sendai virus , which were previously shown to be highly sensitive to RBV treatment To examine whether certain cell types are naturally resistant to RBV even without prior drug exposure, we selected seven different cell lines from various hosts and compared them for the antiviral activities of RBV against two nonsegmented negative-strand RNA viruses (order 2 atmosphere at 37°C. VSV-GFP is a recombinant wild type (wt) VSV (Indiana serotype) encoding GFP as an extra gene between the G and L genes The following seven cell lines were used in this study: Syrian golden hamster kidney fibroblast cells ; human cervical adenocarcinoma cells ; human epithelial lung carcinoma cells , mouse mammary gland adenocarcinoma cells , human epidermal carcinoma cells ; and African green monkey kidney cells . In addition, we used BSRT7 cells which are derived from BHK21, constitutively express bacteriophage T7 polymerase and described by Buchholz et al. S-(4-Nitrobenzyl)-6-thioinosine  from Sigma-Aldrich. Stock solution of RBV (0.1 M) was made in H2O, while ActD (2 mg/ml) was dissolved in 100% ethyl alcohol, and guanosine (20 mM) and NBMPR (16.8 mM) in DMSO.RBV was purchased from MP Biomedicals (cat. no. 196066); guanosine (cat. no. 101907) and actinomycin D (ActD) (cat. no. 10465805) from MP Biomedicals; and Most experiments were conducted using 24-well tissue culture plates and nearly 100% confluent cells treated with drugs in SFM (or mock-treated with SFM) and infected with VSV-GFP or SeV-GFP (or mock-infected with SFM) at MOI of 3 CIU/cell. The MOI for each virus/cell type combination was calculated by infecting each cell line with VSV-GFP or SeV-GFP serial dilutions in SFM and counting infectious foci with the aid of fluorescence microscopy. RBV was added to the cells at 24 h before infection. After absorption of virus for 1 h in the absence of drugs (to rule out an interference of drugs with virus attachment/entry), SFM containing unabsorbed virus was removed, cells were washed three times with phosphate-buffered saline (PBS), and 300 µl/well of SFM with the same concentrations of drugs as in the pretreatment was added to each well. The fluorescence and bright field photographs of cells at 10× magnification were captured 24 h post infection (p.i.) or 48 h p.i. using an Olympus DP70 digital camera mounted on an Olympus IX71 inverted fluorescent microscope and Olympus DP Controller software. To examine effect of RBV on virus production, SFM containing infectious particles was collected 24 or 48 h p.i., and viral titrations were performed in 96-well plate format by infecting BHK21 (for VSV) or Vero cells (for SeV) with serial virus dilutions. For SeV titration, cells were overlaid with 100 µl SFM containing 1.2% Avicel RC-581  as previously described The effect of the exogenously added guanosine on VSV and SeV replication in the presence or absence of RBV was examined using confluent monolayers of cells in 96-well tissue culture plates . Cells were infected with either VSV-GFP or SeV-GFP (or mock-infected with SFM) at MOI of 3 CIU/cell. After 1 h p.i., virus was removed and cells were washed with PBS, and mock-treated or treated with the SFM containing 500 µM RBV or 50 µM guanosine, or RBV together with guanosine. Guanosine was dissolved in DMSO and the final concentration of DMSO in the media added to all wells was 0.25%. The intensity of fluorescent signal at 18 h p.i for VSV and 24 h p.i for SeV was quantified using a Fluorescence Multi-Well Plate Reader CytoFluor 4000  with the standard in built Cyto Fluor filter set (excitation wavelength at 485 and emission wavelength at 530 nm). Values were corrected for background fluorescence by subtracting the values of uninfected cells from the value of each infected well.50 and IC90) for RBV, antiviral screening was conducted by means of a plaque reduction assay using 24-well tissue culture plates. Cells were infected with VSV-GFP or SeV-GFP in SFM (or mock-infected with SFM) at an MOI producing about 100 virus plaques per well on each cell line in the absence of RBV. After absorption of virus for 1 h without RBV (to rule out an interference with virus attachment/entry), SFM containing unabsorbed virus was removed, cells were washed three times with phosphate-buffered saline (PBS), and overlaid with 200 µl/well of SFM containing 1.2% Avicel RC-581 and increasing concentrations of RBV. Cells were then incubated for 24 h (VSV) or 48 h (SeV). Plaques were counted with the aid of fluorescence and bright field microscopy, and the 50% (IC50) and 90% (IC90) inhibitory concentrations were calculated. Initial experiments were done using 0, 200, 500 or 1000 µM of RBV as it was done for virus infections at MOI 3 to determine the range of RBV activity for each virus/cell line combination. After that, all plaque reduction experiments were conducted using different ranges of RBV concentrations to more precisely determine the IC50 and IC90 values. Each of these experiments was performed at least twice (done in duplicates) and plaque numbers represent the mean ± standard deviation of the mean.To estimate the 50% and 90% inhibitory concentrations , and 300 µl of fresh SFM was added to each well. SFM from each well was collected at the specified time intervals, flash frozen at −80°C, and analyzed by titration as described above.3H]RBV uptake experiments were conducted essentially as in 3H]RBV  for 15 minutes in a 5% CO2 atmosphere at 37°C. For the long-term accumulation of RBV, cells (in triplicates) on 12-well plates were washed with PBS and treated with 275 µl of SFM (same medium used for infections but without virus) containing the same concentration of RBV/[3H]RBV (in the absence of NBMPR) as above but incubated for 1 h, 16 h or 24 h. To measure intracellular [3H]RBV, cells were then placed on ice for 5 minutes (to stop an uptake) and washed 3 times with cold PBS. The cells were then trypsinized, pelleted at 200×g for 4 minutes and cell pellets were frozen at −80°C. Nucleotide pool isolation was conducted as described in 3H] accumulation. Cell numbers (from separate plates) were counted by two separate methods. First, cells were trypsinized and cell number was determined using a hemocytometer. Cell numbers were independently confirmed by staining monolayers (from a separate plate) with blue-fluorescent Hoechst 33342 dye (Invitrogen), which selectively stains nuclei. At least 5 random fields were photographed using a fluorescence microscope and DAPI filter and nuclei were then counted. Uptake values were determined by dividing the counts per minute (CPM) by number of cells (CPM/cell) in a 24-well plate. For RBV uptake in the presence of ActD, cells were pretreated with 5 µg/ml ActD for 2 h, media was aspirated (without cell washing), and then RBV uptake assay was conducted as described above.Cell monolayers were prepared exactly as for virus infections using 12- or 24-well tissue culture plates. The RBV). After 15-minute incubation, cells were collected and measured for the level of [3H]RBV uptake normalized to the number of cells as described in 3H]RBV counts presented in A recent study by Ibarra and Pfeiffer (2009) showed that the development of cell-based resistance to RBV treatment via decreased RBV uptake can greatly limit RBV antiviral activity. Therefore, we wanted to examine a possibility that the RBV resistance of Vero, BHK21 and A549 cells was a result of defective RBV uptake in these cell types, using methodology similar to that described previously 3H]RBV, which depends on the RBV metabolism) was different in the seven cell lines. To test it, we conducted a similar uptake experiment described above but with cells treated with [3H]RBV for 1 h, 16 h and 24h (instead of 15 minutes). As shown in While our short-term uptake experiments did not reveal any defects in RBV import in the seven cell lines, we wanted to see whether long-term accumulation of -guanosine uptake (data not shown). The addition of 50 µM guanosine was unable to neutralize the RBV effect in these 4 cell lines even when the RBV concentration was lowered to 200 or 100 µM (data not shown). Also, a similar result was obtained when 200 µM guanosine was added to the medium (data not shown). These data suggest that a decrease in the cellular GTP pool is not the predominant mechanism of RBV action against VSV and SeV in HeLa, 4T1, HEp-2 and BSRT7 cells, and that other mechanisms also contribute to RBV activity against these two viruses in those cell lines.One of the major proposed mechanisms of RBV antiviral action is the inhibition of the host enzyme IMPDH essential for the 3H]RBV uptake experiment conducted as described in Previous studies showed that actinomycin D (ActD), an inhibitor of DNA-primed RNA synthesis , was able to revert the antiviral effect of RBV against several RNA viruses, including VSV As noted, the seven cell lines used in this study were selected solely based on their ability to support replication of VSV and SeV. To assess any possible correlation between the general ability of these viruses to replicate in these cell lines and their resistance to RBV, we compared VSV and SeV for their ability to initiate infection and for their replication kinetics in these cell lines without RBV treatment. First, VSV-GFP or SeV-GFP virus stocks were titrated in parallel on different cell lines and the relative ability of each virus to initiate virus infection was calculated by counting infectious foci generated on each cell line. As shown in We also conducted one-step growth kinetics analysis by infecting each cell type with VSV-GFP or SeV-GFP at MOI of 3 CIU/cell  and measuring production of new infectious particles by collecting medium from each well at specified time points and titrating it as described in Mononegavirales, VSV (a rhabdovirus) and SeV (a paramyxovirus), in seven different cell lines originated from various hosts and tissues. Previous studies showed that RBV can effectively inhibit replication of VSV MononegaviralesMononegavirales, they belong to different families and have noticeably different growth kinetics in these cell lines.In this study, we compared the antiviral activity of RBV against two prototypic members of the order 50 = 2250 µM for VSV and 1550 µM for SeV; IC90>3000 µM for both viruses), moderate activity in BHK21 and A549 cells, and very effective inhibition in HEp2, HeLa, 4T1 and BSRT7 cells . This pattern was confirmed using various infection and RBV treatment conditions, with cells infected and treated at 34 or 37°C, high or low MOI, and with RBV treatment starting at 24 h before infection, 6 h before infection, or 1 h p.i. Using three different cell viability assays, we showed that RBV treatment even at 1000 µM concentration did not produce any significant cytotoxicity in any of the tested cell lines at our experimental conditions, nor did we observe any significant differences between tested cell types, indicating that the observed pattern of RBV resistance was not due to differences in RBV toxicity. It is important to emphasize that the median RBV plasma concentration in HCV patients at the peak of RBV therapy is between 6.6and 9 µM 50 and IC90 values for Vero, BHK21 and A549 cells  neutralizing effect on RBV in BHK21, A549 and Vero cells, which are already highly resistant to RBV. However, very little effect was observed on the RBV activities in RBV-sensitive cells, especially HeLa, 4T1 and HEp-2 cells. Together, these data suggest that a decrease in the cellular GTP pool is not the predominant mechanism of RBV action against VSV and SeV in HeLa, 4T1, HEp-2 and BSRT7 cells, and that other mechanisms also contribute to RBV activity against these two viruses in these cell lines.Unlike guanosine, ActD was able to effectively neutralize RBV in all tested cell lines. Previous studies showed that ActD neutralizes RBV effects via two mechanisms . Malinoski and Stollar (1980) showed that ActD neutralized effect of RBV against Sindbis virus by maintaining the GTP pool size at its normal level (the mechanism of this stabilization is still unknown) Paramyxoviridae, genus Morbillivirus) in Vero cells  Based on the ability of ActD (but not guanosine) to neutralize the effect of RBV in RBV-sensitive cell lines , we hypothesize that RBV antiviral activity in these cell lines depends not only on the depletion of the GTP pool (can be restored by guanosine addition) but also on the successful 5′-phosphorylation of RBV into RMP/RDP/RTP At present, we cannot explain dramatic differences between BHK21 and BSRT7 cells in their resistance to RBV and the long-term RBV accumulation. BSRT7 cell line is derived from BHK21 and constitutively express bacteriophage T7 polymerase under control of the cytomegalovirus promoter and the neomycin resistance gene Respirovirus) inhibition by RBV has not been previously studied, a previous study on RSV  suggests the predominant mechanism of action of RBV against RSV is inhibition of cellular IMPDH activity by RMP (and consequent decrease in the cellular GTP pool) rather than interactions of RBV metabolites with the viral polymerase We believe the very similar pattern of RBV activity against VSV and SeV in seven different cells lines may indicate that these two viruses are inhibited by RBV via the same mechanism. Although the mechanism of SeV  against important viral pathogens. Future experiments are also needed to determine whether the observed differences between different cell lines are limited only to nonsegmented negative-strand RNA viruses by analyzing effect of RBV on replication of positive-strand RNA or segmented negative-strand RNA viruses in these cell lines. Finally, our results strongly point out the importance of using multiple cell lines of different origin when antiviral efficacy and potency are examined for new as well as established drugs in vitro.


Proliferating cell nuclear antigen (PCNA) is a nuclear protein that regulates DNA synthesis by DNA polymerase delta, and is essential for DNA replication. PCNA expression level is related to the malignancy of gastric cancer cells. Seven different gastric cancer cell lines and two kinds of control cell lines were treated with antisense oligonucleotides complementary to the messenger RNA of PCNA. Treatment of each gastric cancer cell line with antisense oligonucleotides at concentration of 10-40 microM inhibited the cell growth, colony formation and PCNA protein production in a dose-dependent manner, but only affected normal cells slightly. A random sequence oligomer showed no effect. These results show that PCNA is essential for gastric cancer cell proliferation and that the use of synthetic oligonucleotides is an effective way of producing antisense-mediated changes in the behaviour of human gastric cancers.


Two tumour-cell-aggregation factors, derived from rat ascites hepatoma cells, had different antigenicity; one was not absorbed by immunoadsorbent chromatography with anti-rat serum antibody and the other was. Their activities were both lost by digestion with trypsin, but remained unchanged by oxidation with periodate, suggesting the role of the protein portions in their molecules. The potency of the unabsorbed factor was inhibited specifically by alpha-methyl-D-mannoside or D-mannose, while that of the absorbed factor was inhibited specifically by N-acetyl-D-glucosamine, suggesting that these carbohydrates may be concerned with the respective receptor structures at the tumour-cell surface. The unabsorbed factor induced not only cell aggregation (as shown in the form of simple apposition) but also cell adhesiveness characterized by development of intermediate junctions, desmosomes and tight junctions, while the absorbed factor produced only simple apposition, suggesting their functional difference.


The effect of basement membrane components  and lung fibroblasts on type IV collagenase and plasminogen activator activity was investigated in a primary HSV-2-induced hamster fibrosarcoma, and its in vivo derived sublines and in vitro derived clones of varying metastatic potential. Fibronectin and type IV collagen were ineffective at influencing the expression of either type IV collagenase or plasminogen activator activity. Laminin, however, at concentrations of 1-10 micrograms ml-1 added to the serum-free culture supernatants, increased the release of type IV collagenase by up to 100% for the parental cell line. Three highly metastatic sublines (two from in vivo origin and one from in vitro cloning) showed increases of up to 300%. Non-metastatic sublines (two from in vivo origin and one from in vitro cloning), however, showed no increase in type IV collagenase activity. Plasminogen activator release from either the parental line cell or its metastatic sublines and clones, was unaffected by the addition of laminin. Addition of tumour cells to lung fibroblast monolayers resulted in an increased expression of PA activity in the supernatant, whilst type IV collagenase activity was reduced.


The effects of various chemotherapeutic agents on alpha-fetoprotein (AFP) secretion and growth of human hepatocellular carcinoma and hepatoblastoma cell lines were investigated in vitro. It was found that there was a high correlation between hepatoma cell number and AFP secretion after treatment and that the amount of AFP secreted per cell per 72 h was not affected with therapeutically achievable concentrations. These results suggest that serum AFP level in patients with hepatomas does not correlate with the size of whole tumour but with that of viable tumour mass, and that AFP-secreting capacity of tumour cells in the mass is kept unchanged after chemotherapy.


Cholangiocarcinoma is a serious public health in Thailand with increasing incidence and mortality rates. The present study aimed to investigate cytotoxic activities of crude ethanol extracts of a total of 28 plants and 5 recipes used in Thai folklore medicine against human cholangiocarcinoma (CL-6), human laryngeal (Hep-2), and human hepatocarcinoma (HepG2) cell lines in vitro.50 (concentration that inhibits cell growth by 50%) and the selectivity index (SI) were calculated.Cytotoxic activity of the plant extracts against the cancerous cell lines compared with normal cell line  were assessed using MTT assay. 5-fluorouracil was used as a positive control. The ICAtractylodes lancea, Kaempferia galangal, Zingiber officinal, Piper chaba, Mesua ferrea, Ligusticum sinense, Mimusops elengi) and one folklore recipe (Pra-Sa-Prao-Yhai) exhibited promising activity against the cholangiocarcinoma CL-6 cell line with survival of less than 50% at the concentration of 50 μg/ml. Among these, the extracts from the five plants and one recipe  showed potent cytotoxic activity with mean IC50 values of 24.09, 37.36, 34.26, 40.74, 48.23 and 44.12 μg/ml, respectively. All possessed high activity against Hep-2 cell with mean IC50 ranging from 18.93 to 32.40 μg/ml. In contrast, activity against the hepatoma cell HepG2 varied markedly; mean IC50 ranged from 9.67 to 115.47 μg/ml. The only promising extract was from Zingiber officinal (IC50 = 9.67 μg/ml). The sensitivity of all the four cells to 5-FU also varied according to cell types, particularly with CL-6 cell (IC50 = 757 micromolar). The extract from Atractylodes lancea appears to be both the most potent and most selective against cholangiocarcinoma .The extracts from seven plant species (The ethanolic extracts from five plants and one folklore recipe showed potent cytotoxic activity against CL-6 cell. Sensitivity to other cancerous cell lines varied according to cell types and the hepatocarcinoma cell line. HepG2 appears to be the most resistant to the tested extracts. Authentication of plant materials was carried out at the herbarium of the Department of Forestry, Bangkok, Thailand, where the herbarium vouchers have been kept. A duplicate set has been deposited in the herbarium of Southern Center of Thai Medicinal Plants at the Faculty of Pharmaceutical Science, Prince of Songkhla University, Songkhla, Thailand.The plant materials were rinsed thoroughly with tap water to remove extraneous contaminants and cut into small pieces, oven-dried at 50°C until stability of dry weight was observed, and then ground into powder with an electric-grinder. Extraction was carried out by macerating the powdered plant materials (100 g) in stoppered flasks containing 500 ml of 95% ethanol at room temperature (25-30°C) for 7 days. The extracted solvent was separated and filtered through Whatman no. 1 filter paper. After filtration, the extracts were evaporated under reduced pressure by rotary evaporation. The crude extracts were weighed and stored at −20°C until used.The crude extract from each medicinal plant/recipe was initially dissolved in 50% ethanol. Concentrated stock solution of each extract was prepared by adding a known weight of each crude extract to a known volume of 50% ethanol, and then serially diluted (1:2) with complete media to obtain the working solutions at eight final concentrations. Positive control agent 5-FU was prepared similarly as the crude extract by dissolving in 50% ethanol.2 atmosphere with 95% humidity.The cholangiocarcinoma cell line CL-6, human laryngeal carcinoma cell line Hep-2, human hepatocarcinoma cell line HepG2 and normal human epithelial cell (HRE) were used for cytotoxic screening of the medicinal plant extracts. CL-6 cell line was established and kindly provided by Associate Professor Dr. Adisak Wongkajornsilp, Department of Pharmacology, Faculty of Medicine , Mahidol University, and were cultured in Ham-12 medium supplemented with 10% heated fetal bovine serum and 100 IU/ml of antibiotic-antimycotic solution. Hep-2 cell line was obtained from Department of Medical Technology, Faculty of Allied Health Sciences, Thammasat University, established and cultured in M-199 medium supplemented with 10% heated fetal bovine serum, 2.5 mM HEPES (pH 7.4) and 100 IU/ml penicillin-streptomycin solution. HepG2 cell line was purchased from the Cell Line Service Co. Ltd. (Germany) and was cultured in DMEM: Ham's F12 medium supplemented with 2 mM L-glutamine, 10% fetal bovine serum and 100 IU/ml pen-strep. Normal human renal epithelial cell line (HRE) was purchased from Promocell Co. Ltd. (Germany) and cultured in renal epithelial cell growth medium 2 with SupplementPack. All cells were maintained at 37°C in a 5% CO4 cells/well in 100 μl culture medium. Following 24-h incubation and attachment, the cells were treated with different concentrations of plant extracts and 5-FU (positive control) for 24 h. Each extract was screened initially for its cytotoxicity against all cancerous and normal cell lines at the concentration of 50 μg/ml. The potential candidates which resulted in cell survival of less than 50% were further assessed for their IC50 (concentration that inhibits cell growth by 50%) values at the concentration range of 250 and 1.95 μg/ml. The concentration range used for 5-FU was 78.13 to 10,000 μM. Following washing and incubation with MTT solution (20 μl of 5 mg/ml) at 37°C for 3 h, cells were lyzed with DMSO. The yellow MTT dye was reduced by succinic dehydrogenase in the mitochondria of viable cells to purple formazan crystals. Absorbance (OD) was measured at 570 nm using a microplate reader . The percentage of cytotoxicity compared to the untreated cells was determined with the equation:The MTT colorimetric assay developed by Mosmann  with mod50 values were calculated using CalcySyn™ (USA) software. The selectivity index (SI) was also calculated from the IC50 ratio of normal epithelial and cancerous  cells. SI value indicates selectivity of the sample to the cell lines tested. Samples with SI value greater than 3 were considered to have high selectivity.The results were generated from three independent experiments; each experiment was performed in triplicate. The ICBridelia ovata, Curcuma zedoaria, Derris scandens, Dioscorea membranacea, Nardostachys jatamansi and Rhinacanthus nasutus) showed promising cytotoxic activity (IC50 < 30 μg/ml)   . The alcro cells . In the 35.72 μM . The actAtractylodes lancea, Kaempferia galangal, Zingiber officinal, Piper chaba, Mesua ferrea, and Pra-Sa-Prao-Yhai recipe) used in Thai folklore medicine exhibited promising cytotoxic activity against CL-6 human cholangiocarcinoma cell line. Sensitivity to other cancerous cell lines varied according to cell types and the hepatocarcinoma HepG2 appears to be the most resistant cell line to the tested extracts. The extract from Atractylodes lancea appears to be both the most potent and most selective against cholangiocarcinoma, whereas that from Zingiber officinal appears to be the most potent and most selective against HepG2. The extract from Piper chaba  and Pra-Sa-Prao-Yhai recipe  exhibited the most promising and most selective cytotoxic activity against Hep-2 cell line. Further investigation of all the six extracts for their cytotoxic activity against cholangiocarcinoma in hamster model is underway to fully assess the anticancer activity in vivo.Results obtained from this study indicate that 6 out of a total of 28 plants and 5 recipes (The authors declare that they have no competing interests.KN conceived and designed the study, reviewed and finalized the manuscript. WM performed the laboratory work, and drafted the manuscript. VV obtained the financial support for the project, reviewed and provided comments and suggestions to improve the quality of the manuscript. WC performed data analysis. AI prepared the medicinal plants and all the herbal extracts. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1472-6882/10/55/prepub


The interaction of normal (CHO-K1) and multidrug-resistant (Adrr) Chinese hamster ovary cells was examined in mixed monolayer and spheroid culture. In order to assess the individual response of the two cell types in mixed culture, CHO-K1 was genetically marked by transfection with a bacterial beta-galactosidase gene. The enzyme product can be detected histochemically and allows identification of the marked cell line, designated CHO-K1-BG. Following administration of doxorubicin or mitozantrone, there was a large difference in the clonogenic survival of CHO-K1-BG and Adrr, whereas the overall survival of a 50:50 mixture of the two cell lines had intermediate values. When the survival of marked and unmarked colonies from mixed culture was assessed separately, there was no detectable alteration in chemosensitivity. We have found no evidence for interaction of sensitive and multidrug-resistant cells in this system.


The RAS/RAF/MEK/ERK pathway is involved in the balance between melanocyte proliferation and differentiation. The same pathway is constitutively activated in cutaneous and uveal melanoma (UM) and related to tumour growth and survival. Whereas mutant BRAF and NRAS are responsible for the activation of the RAS/RAF/MEK/ERK pathway in most cutaneous melanoma, mutations in these genes are usually absent in UM.We set out to explore the RAS/RAF/MEK/ERK pathway and used mitogen-activated protein kinase profiling and tyrosine kinase arrays.We identified Src as a kinase that is associated with ERK1/2 activation in UM. However, low Src levels and reduced ERK1/2 activation in metastatic cell lines suggest that proliferation in metastases can become independent of Src and RAS/RAF/MEK/ERK signalling. Inhibition of Src led to the growth reduction of primary UM cultures and cell lines, whereas metastatic cell line growth was only slightly reduced.We identified Src as an important kinase and a potential target for treatment in primary UM. Metastasis cell lines seemed largely resistant to Src inhibition and indicate that in metastases treatment, a different approach may be required. CDKN2A in CM, most of the UM cell lines posses a wild-type p16-encoding gene that is, however, not expressed because of the epigenetic modification of the CDKN2A gene (NRAS (20% of cases) and BRAF (60% of cases) genes  is a rare neoplasm that arises from melanocytes in the eyes. It usually affects people in their sixties with an incidence rate of ∼6–8 new cases per million per year among Caucasians . Little M L-glutamine (Gibco), 2% penicillin/streptomycin and 10% FBS . Primary UM was cultured in Amniochrome Pro Medium . All cell cultures were incubated at 37°C in a humidified 5% CO2 atmosphere.A total of 11 cell lines derived from primary UM  and UM metastases  were analysed for kinase activity , supplemented with 1% Halt Protease Inhibitor Cocktail, EDTA-free (Pierce) and 1% Halt Phosphatase Inhibitor Cocktail (Pierce). Protein concentrations were measured by using the BCA Protein Assay kit (Pierce). Cell lysates were also acquired from three fresh primary UM samples obtained by enucleation and from three liver metastases of three different patients, in whom the diagnosis was confirmed.The Human Phospho-MAPK Array  was used to simultaneously detect the relative levels of nine MAP kinases and nine other serine/threonine kinases in cell lines, in primary UM and in liver metastasis. In this array, capture and control antibodies were spotted in duplicate on nitrocellulose membranes. Experiments were carried out according to the manufacturer's guidelines. In short, cell lysates were diluted and incubated with the array. After binding of both phosphorylated and unphosphorylated kinases, unbound material was washed away. A cocktail of phospho-site-specific biotinylated antibodies was used to detect phosphorylated proteins through streptavidin-horseradish peroxidase and chemiluminescence. X-ray films of the blots were scanned and analysed using G-boxHR . Control spots with mouse, goat and rabbit antibodies were used for background correction.μg cell lysate, 4 μl 100 × BSA (New England Biolabs), 0.4 μl 10 mM ATP  and 0.5 μl 1 mg ml−1 monoclonal anti-phosphotyrosine FITC conjugate , adjusted to 40 μl with distilled H2O. The sample was pulsed back and forth through the porous material for 45 cycles, which is coupled to the base of a well to maximise reaction kinetics and to reduce analysis time. At every fifth pump cycle, a 16-bit TIFF image was taken with a built-in CCD camera (PamGene).Experiments were carried out using a 4-array semi-automated system  designed for processing PamChip-4 arrays. The PamChip Tyrosine Kinase Array (PamGene) contains 144 phospho-peptides, immobilised on a porous microarray surface through the peptide N terminus, representing tyrosine kinase substrates. Each array was blocked with 0.2% bovine serum albumin (BSA), fraction V  by pumping it through the porous microarray for 30 cycles of 30 s. Thereafter, each array was washed thrice for 8 s with 1 × ABL Protein Tyrosine Kinase Reaction Buffer solution . Next, incubation was carried out at 30°C with the reaction mix, containing 5 μM in line with a large body of literature. Each particular inhibitor was mixed with lysates of cell lines and tissue, together with the reaction mix just before incubation on the array.Blocking experiments were carried out with Src family-selective tyrosine kinase inhibitors, PP1, PP2  and PP3 , at an end concentration of 10 P-values of 0.05 or less were corrected for multiple testing using the Benjamini and Hochberg correction. Substrates with a corrected P-value of 0.05 or less were assumed to be significant.Acquired data from the PamStation 4 were captured with the supplied software package BioNavigator . For the purpose of finding differentially phosphorylated substrates, data were imported in the LIMMA package (Bioconductor.org) and we applied the empirical Bayes method . Backgroμg) were separated on 12.5% SDS–PAGE gels, and proteins were transferred to Hybond-polyvinyldifluoride membranes . The membranes were blocked with 5% skim milk in a PBS-Tween 0.1% solution and probed at room temperature for 1 h with antibodies specific to each antigen: phospho-Src , phospho-Src family  and Src  antibody . An antibody against actin  was used as a loading control. Membranes were subsequently incubated at room temperature with horseradish peroxidase-conjugated IgG anti-mouse or anti-rabbit secondary antibodies for 1 h. Supersignal West Femto ECL (Pierce) was used to visualise protein bands on the membrane.Cell lysates (10 M) were incubated in the standard medium with reduced serum (1%), according to the manufacturer's instructions. The siRNA constructs  were predesigned and validated (∼70% knockdown) by the manufacturer (Invitrogen). After 24 and 48 h, the cells were harvested and RNA and protein lysates were prepared.Sub-confluent cell cultures were grown without antibiotics 24 h before transfection in RPMI 1640 medium. A mixture of Lipofectamine 2000  and two different siRNA constructs (40 nμM) was measured by mitochondrial function using the WST-1 proliferation reagent (Sigma-Aldrich) as described earlier . Primer sequences for Src: 5′-GCTGCGGCTGGAGGTCAAG-3′ (forward) and 5′-AGACATCGTGCCAGGCTTCAG-3′ (reverse). Primer sequence for β-actin: 5′-CGGGACCTGACTGACTACCTC-3′ (forward) and 5′-CTCCTTAATGTCACGCACGATTTC-3′ (reverse). The PCR reaction settings were 95°C for 5 min, then 40 cycles at 96°C for 15 s and at 60°C for 45 s. The DNA melting point of the amplicons was acquired by measuring the fluorescence of SYBR Green  during a linear temperature transition from 70 to 97°C at 0.2°C each for 10 s with accompanying software (Bio-Rad).The cell lines  were analysed for Src gene expression. Primers for Src and the reference gene, An antibody array was applied to investigate the MAPK pathway in 10 UM cell lines, in three primary UM and three UM metastasis. We observed a uniform HSP27 phosphorylation, with the exception of in three UM cell lines . UMs displaying activated ERK1/2 as well as phosphorylated HSP27 were most common, whereas signals for phosphorylated ERK1/2 were low in metastasis tissue (MET1-3) and metastatic UM cell lines  . RemarkaBRAF and NRAS mutations.Reduction of ERK1/2 activation in metastatic cell lines compared with that in primary UM cell lines provides a model to identify the underlying mechanism of ERK1/2 activation in the absence of To investigate whether a kinase is differentially activated between primary UM cell lines and metastatic UM cell lines, we used peptide-based tyrosine kinase arrays . After nin vitro inhibition experiments with the Src-kinase-specific inhibitors PP1 and the PP1 analogue, PP2. We added PP1 and PP2 (10 μM) to lysates of primary UM tissue and of a primary UM cell line and measured the inhibitory effect of these Src inhibitors using kinase array  resulted in a decimation of kinase activity similar to the inhibition that we observed in UM tissue  and 527 (Y527). The expression of phosphorylated Src Y416, which is associated with an active conformation, was low in the metastatic UM cell lines . SurprisTo investigate the origin of a lowered Src expression, we performed a gene expression analysis . Src genA western analysis of Src expression in UM and metastasis tissue revealed a very high Src expression in only one out of three primary UM, whereas all three metastasis tissues displayed medium expression of Src protein .in vitro. However, in endometrial and breast cancer, ERK1/2 activation has been associated with a good prognosis (Constitutive activation of ERK1/2 has often been reported for UM (BRAF and NRAS) are lacking (BRAF V600E (OCM1) and GNAQ Q209L (Mel202) mutation status (Metastatic and UM cell lines provide a unique model to identify the mechanisms that regulate ERK1/2 activation in UM. Earlier work already showed that ERK1/2 phosphorylation in UM depends on the MAPK pathway, although mutations in the usual suspects (e.g.,  lacking . To specIn conclusion, we have identified a differential ERK1/2 activation in UM and metastatic UM cell lines. Using tyrosine kinase activity profiling, we identified Src as a determinant of ERK1/2 activation and showed that Src expression and kinase activity, together with ERK1/2 activation, are reduced in UM metastases cell lines.


The acute sensitivity of some hybridoma cell lines to culture-related stresses severely limits their productivity. Recent developments in the characterization of the stress signals modulating the cellular phenotype revealed that the pro-apoptotic transcription factor Gadd153 could be used as a marker to facilitate the optimization of mammalian cell cultures. In this report, we analyzed the expression of Gadd153 in Sp2/0-Ag14 murine hybridoma cells grown in stationary batch culture and subjected to two different culture optimization paradigms: L-glutamine supplementation and ectopic expression of Bcl-xL, an anti-apoptotic gene.The expression of Gadd153 was found to increase in Sp2/0-Ag14 cells in a manner which coincided with the decline in cell viability. L-glutamine supplementation prolonged Sp2/0-Ag14 cell survival and greatly suppressed Gadd153 expression both at the mRNA and protein level. However, Gadd153 levels remained low after L-glutamine supplementation even as cell viability declined. Bcl-xL overexpression also extended Sp2/0-Ag14 cell viability, initially delayed the induction of Gadd153, but did not prevent the increase in Gadd153 protein levels during the later phase of the culture, when cell viability was declining. Interestingly, L-glutamine supplementation prevented Gadd153 up-regulation in cells ectopically expressing Bcl-xL, but had no effect on cell viability.This study highlights important limitations to the use of Gadd153 as an indicator of cell stress in hybridoma cells. Mammalian cell lines provide several advantages over other cellular systems for the production of recombinant proteins, most notably the correct processing and modification of mammalian proteins . UnfortuRecent efforts have also focused on the characterization of the stress-induced signaling pathways leading to changes in the cellular phenotype ,6. Of paThe mouse hybridoma Sp2/0-Ag14 (Sp2/0) cell line offers several advantages for studying the regulation of hybridoma cell viability. Firstly, this cell line is acutely sensitive to culture-related stresses. In particular, Sp2/0 cells rapidly undergo apoptosis after 4 days of stationary batch culture due to L-glutamine depletion . Thus, tIn this report, we studied the regulation of Gadd153 in wild type Sp2/0 cells and in Sp2/0 cells overexpressing Bcl-xL. Our results show that, while Gadd153 is tightly regulated by L-glutamine in both cell lines, Gadd153 protein levels remained low after L-glutamine supplementation, even as cell viability progressively decreased. Furthermore, reducing Gadd153 levels by L-glutamine supplementation in the Bcl-xL-expressing cell line did not impact on cell viability. This indicates that the usefulness of Gadd153 as a marker of the health of mammalian cell cultures is limited.We first analyzed the regulation of Gadd153 protein levels in Sp2/0 cells grown under stationary batch culture conditions. Gadd153 expression was very low during the first 4 days of culture, which corresponded to the lag and exponential phases of cell growth Fig.  and 1B. For Gadd153 to be useful to monitor the stress level of hybridoma cultures, its expression must be responsive to strategies that improve cell viability. Therefore, we investigated the impact of L-glutamine supplementation on the expression of Gadd153 in Sp2/0 stationary batch cultures. In agreement with our previous observations , L-glutaThe viability of Sp2/0 cultures can also be improved by the ectopic expression of the anti-apoptotic protein Bcl-xL ,18. We tWe next investigated the expression of Gadd153 upon L-glutamine supplementation of Bcl-xL-Sp2/0 cultures. L-glutamine supplementation on culture day 4 lead to a significant increase in cell viability for the pTEJ8-Sp2/0 control cell line Fig.  and 4B. The optimization of mammalian cell culture processes would greatly benefit from the availability of a cellular marker that would 1) accurately reflect the health of the culture and 2) respond to strategies aimed at improving cell viability. Because its expression is induced by several stresses encountered during the large scale culture of mammalian cells, the pro-apoptotic transcription factor Gadd153 was recently proposed to be an indicator of apoptosis occurring in cell cultures ,14.To validate the suitability of Gadd153 as a stress marker for hybridomas, we studied its expression in Sp2/0 cells. Even though it does not secrete immunoglobulins , its higWe also examined the regulation of Gadd153 under two conditions known to improve the behavior of the Sp2/0 cell line in batch culture: L-glutamine supplementation and the ectopic expression of Bcl-xL. We observed that Gadd153 mRNA and protein levels were significantly decreased in Sp2/0 cultures supplemented with L-glutamine Fig. . This reOur data also revealed that the reduction in Gadd153 protein expression upon L-glutamine-supplementation persisted for the rest of the culture period, even as cell viability continued to decrease. This indicates that supplementing the culture with L-glutamine at the peak of Sp2/0 cell viability is sufficient to override further increases in Gadd153 protein levels, whatever the nature of the stresses leading to the loss of Sp2/0 cell viability. This phenomenon was not observed in other studies involving batch cultures because of the nature of the experimental design. Effectively, while Murphy et al.  limited We extended our studies to the use of Sp2/0 cells engineered to express high levels of the anti-apoptotic protein Bcl-xL. This manipulation was shown previously to significantly improve the behavior of Sp2/0 cells in batch culture ,18. InteThe pro-apoptotic protein Gadd153 is acutely up-regulated by several stresses encountered during the large scale culture of mammalian cells. As such, its use as an indicator of cell stress can facilitate the optimization of cell cultures. However, our data highlight important limitations to the use of Gadd153 as a stress marker in mammalian batch cultures. Whether these limitations will be encountered in other cell lines or when other feeding mixtures or culture optimization strategies are used remains to be determined.2HPO4, 1.7 mM NaH2PO4, 150 mM NaCl, pH 7.4) and adjusted to pH 7.2.Unless stated otherwise, all reagents were from Sigma-Aldrich . L-glutamine was prepared fresh as 200 mM stock solutions in phosphate buffered saline  and cultured in Iscove's Modified Dulbecco's Media (IMDM) supplemented with 5% Fetal Clone , 100 U/mL penicillin, 100 μg/mL streptomycin and 4 mM L-glutamine (hereafter referred to as complete IMDM). The pTEJ8- and pTEJ8-Bcl-xL-transfected Sp2/0 cells were described previously  and were4 cells/mL. Samples were then taken at the indicated time intervals for the determination of cell viability using the trypan blue dye exclusion assay, and for total RNA and protein isolation. Culture supplementation with L-glutamine  was done at the day where the maximal number of viable cells was observed in the controlled cultures, which is on culture day 4 for wild type Sp2/0 and pTEJ8-Sp2/0 cells and day 5 for the Bcl-xL-Sp2/0 transfectants [Stationary batch culture and nutrient supplementation was done as described . Brieflyfectants .NM 008084): 5'ATGGTGAAGGTCGGTGTGAACGGA3' (sense primer) and 5'TTACTCCTTGGAGGCCATGTAGGC3' (antisense primer); Gadd153 (Genebank X67083): 5'ATGGCAGCTGAGTCCCTGCCT3' (sense primer) and 5'TCACATGCTTGGCGCTGGCGC3' (antisense primer). Samples were taken at cycles 21, 24, 27 and 30, and the amplicons were analyzed by agarose gel electrophoresis and ethidium bromide staining.Total RNA isolation was performed as previously described . Reverseet al.[Protein extracts were prepared using a urea-based lysis buffer , according to a procedure described by Shah et al.. ProteinStatistical significance was determined using a one-way analysis of variance and Scheffe's post-hoc test.MM did the majority of the experiments of Figs.


Ras is frequently mutated in cancer, and novel therapies are being developed to target Ras signalling. To identify non-invasive surrogate markers of Ras activation and inhibition, we used31P magnetic resonance spectroscopy (MRS) and investigated NIH 3T3 cells compared to a mutant ras transfected counterpart. The MR spectra indicated that phosphocholine (PC) levels increased significantly from 3 ± 2 fmol cell−1in NIH 3T3 cells to 13 ± 4  fmol cell−1in the transfected cells. The PC/NTP ratio increased significantly from 0.3 ± 0.1 to 0.7 ± 0.3. This could not be explained by either a faster proliferation rate or by alterations in cell cycle distribution. Both cell lines were treated with simvastatin, 17-AAG and R115777, agents which inhibit Ras signalling. Cell proliferation was inhibited in both cell lines. The spectrum of NIH 3T3 cells was not affected by treatment. In contrast, in the ras transfected cells growth inhibition was associated with an average 35 ± 5% drop in PC levels and a comparable drop in PC/NTP. Thus the MRS visible increase in phosphocholine is associated with Ras activation, and response to treatment is associated with partial reversal of phosphocholine increase in ras transfected cells. MRS might therefore be a useful tool in detecting Ras activation and its inhibition following targeted therapies. © 2001 Cancer Research Campaign http://www.bjcancer.com


The structure of [Ph2(HyFoSc)Sn] was confirmed by single-crystal X-ray crystallography. The doubly deprotonated ligand is coordinated to the tin atom through the enolic-oxygen, the azomethine-nitrogen, and phenolic-oxygen, and so acts as an anionic tridentate ligand with the ONO donors. Two carbon atoms complete the fivefold coordination at the tin(IV) center. Intermolecular hydrogen bonding, C–H → π, and π → π interactions combine to stabilize the crystal structure. Compounds 1 and 2 have been evaluated for antiproliferative activity in vitro against the cells of three human tumor cell lines: MCF-7 (human breast cancer cell line), T24 (bladder cancer cell line), A549 , and a mouse fibroblast L-929 cancer cell line.The novel diphenyltin(IV) compound [Ph Organotin compounds are of interest in view of their considerable structural diversity . IncreasThio- and semicarbazones (TSC) possess a wide range of bioactivities, and their chemistry and pharmacological applications have been extensively investigated. The more significant bioactivities of a variety of semicarbazones  and thiosemicarbazones  and their metal complexes have been reviewed together with proposed mechanisms of action and structure-activity relationships , 8. Casa2O with 3-hydroxy-2-formylpyridine semicarbazone H2HyFoSc (1) are described with the final goal of developing new biologically active pharmaceuticals. The results of the cytotoxic activity of 1, SnPh2O, and of the organotin compound (2) against the cells of three human cancer cell lines: MCF-7 (human breast cancer cell line), T24 (bladder cancer cell line), A549 , and a mouse fibroblast L-929 cancer cell line are also reported. To our knowledge, this is the first report of synthesis of 1 and 2.Following our interest in the chemistry and pharmacological properties of thiosemicarbazones –17 and t−1) and nujol mulls dispersed between polyethylene disks (400–40 cm−1). The 1H, 13C NMR spectra were recorded on a Bruker AC-300 MHz and on a Varian 600 MHz spectrometer. The spectra were acquired at room temperature (298 K). The chemical shifts are reported in ppm with respect to the references  for 1H and 13C NMR). Elemental analyses were carried out by the microanalytical service of the University of Ioannina, Greece.The reagents  were used as supplied while the solvents were purified according to standard procedures. Melting points were determined in open capillaries and are uncorrected. Infrared and far-infrared spectra were recorded on a Perkin–Elmer Spectrum GX FT IR System spectrophotometer using KBr pellets  at 80°C for 2 h. Then, the mixture was kept in a refrigerator overnight. The resulting yellow powder was filtered off and recrystallized from EtOH. The powder was washed with cold EtOH and dried in vacuo over silica gel at 40–50°C for 4 h to afford 1 as a yellow powder, yield 75%, and m.p. 230°C. UV-Vis for 1 (DMF) λ/nm (logε): 383 (2.78); 330 sh (3.48); 320 (3.50). IR cm−1: 3275 m, 3208 m (ν(OH)); 3147 s, 3080 m, 2920 ); 1664 (ν(C=O)), 1583 s (ν(C=N)); 1322 (ν(C–O)); 916 s (ν(NN)). 1H-NMR (DMSO-d6): δ 12.36 , 11.23 , 8.05 , 7.80 , 8.35 , 8.14 , 6.75 ; 13C-NMR: δ 131.4 (C2), 154.0 (C3), 126.4 (C4), 126.4 (C5), 133.70 (C6), 134.7 (C7), 155.9 (C8=O). Anal. calc. for C7H8N4O2 (180.0 g mol−1): C 46.7, H 4.5, N 31.10; found: C 46.6, H 4.3, N 31.3%. Commercially available 3-hydroxy-(2-hydroxymethyl)pyridine hydrochloride was oxidized with MnOrding to , 23 to a2O (Dean–Stark trap). The resulting clear solution was concentrated in vacuo to a small volume. The oily product was chilled and triturated with distilled diethyl ether (Et2O) to give a yellow solid. The yellow powder was recrystallized from distilled ether and was dried in vacuo over silica gel. Yield 24%; m.p. 209°C. UV-Vis for 2 (DMF) λ/nm (logε): 388br (3.87); 331 sh (4.15); 320 (4.18). IR cm−1: 3144s, 3065 m, (ν(NH2)); 1664 (ν(C=O)), 1542 s (ν(C=N)); 1283 (ν(C–O)); 997 s (ν(NN)) 339 sh, 321 ms (ν(SnC)); 446 sh (ν(SnNC=N)); 284 mw (ν(SnOC=O)); 248 mw (ν(SnO)). 1H-NMR (DMSO-d6): δ 7.30 , 7.36 , 8.15 , 8.20 , 6.74 , 7.95 , 7.45, 7.57 . 13C-NMR: δ 137.5 (C2), 153.2 (C3), 128.0 (C4), 128.2 (C5), 138.0 (C6), 139.5 (C7), 155.8 (C8=O), 134.7 (Co), 127.7 (Cm), 127.2 (Cp). Anal. calc. for C19H16N4O2Sn (451.0 g mol−1) C, 50.6; H, 3.6; N, 12.4; Found: C, 50.4; H, 3.7; N, 12.3%. Crystals suitable for X-ray analysis were obtained by slow evaporation of a freshly distilled diethyl ether solution of 2.Diphenyltin(IV) oxide  and 3-hydroxypyridine-2-carbaldehyde semicarbazone  in benzene (100 mL) were refluxed for 24 h under azeotropic removal of Hα radiation (λ = 0.71073 Å). The data were corrected for Lorentz and polarization effects. An analytical absorption correction was applied to the data using a multifaceted crystal model  or e-mail mdeposit@ccdc.cam.ac.uk or http://www.ccdc.cam.ac.uk.Crystallographic data for μL of DMSO completed with 900 μL of tissue culture medium. Afterwards, the tested compounds were diluted in culture medium to reach the final concentrations of 100, 50, 10, 1, and 0.1 ng/μL. The solvent (DMSO) in the highest concentration used in the test did not reveal any cytotoxic activity.Test solutions of the tested compounds (1 mg/mL) were prepared by dissolving the substance in 100 4 cells per well. The MCF-7 cells were cultured in the D-MEM (Modified Eagle's Medium) medium supplemented with 1% antibiotic and 10% fetal calf serum. L-929 cells were grown in Hepes-buffered RPMI 1640 medium supplemented with 10% fetal calf serum, penicillin (50 U/mL), and streptomycin (50 mg/mL). A-549 cells were grown in F-12K Ham's medium supplemented with 1% glutamine, 1% antibiotic/antimycotic, 2% NaHCO3, and 10% fetal calf serum. The cell cultures were maintained at 37°C in a humid atmosphere saturated with 5% CO2. Cell numbers were counted by the Trypan blue dye exclusion method. MCF-7, L-929, and A-549 cells were determined by the sulforhodamine B assay . Th. Th1 was2, together with the atom-labelling scheme, is given in A perspective view of τ = 0.54 . The metal coordination geometry is therefore described as distorted trigonal bipyramidal with the O(2) and O(1) atoms occupying the apical positions around the tin atom.The doubly deprotonated ligand is coordinated to the tin atom through the enolic-oxygen, azomethine-nitrogen, and phenolic-oxygen atoms. Two carbon atoms complete the fivefold coordination at the diorganotin(IV) fragment. Analysis of the shape determining angles using the approach of Addison et al.  yields τZEZ configuration, The dianionic, tridentate ONO ligand has a 2 are connected into dimers by a pair of cooperative hydrogen bonds [see π and π→π contacts further stabilizes the crystal structure.The polar hydrogen atoms on N(3) participate in two intermolecular hydrogen bonds. The monomers of 2HyFoSc (1), the strong bands at 3147 and 3088 cm−1 are assigned to the asymmetric and symmetric modes of terminal NH2, respectively. The ν(NH) band appears at 2920 cm−1. The strong broad band at ca. 2650 cm−1 is assigned to the ν(NH ⋯ O) and ν(OH ⋯ O) mode due to strong intra- or intermolecular hydrogen bonding. The absence of the ν(NH) stretching motion at 2 is indicative of deprotonation of the amide proton. The strong broad band at ca. 2650 cm−1 at 2 is probably due to the ν(NH ⋯ N) intermolecular hydrogen bonds as confirmed by X-ray crystallography. The coordination of the azomethine-N atom to the tin center was suggested in the IR spectrum by a shift of the ν(C=N) band to a lower frequency, along with the occurrence of a ν(N–N) band to higher frequency [−1 for 1 was assigned to ν(C–O). This band was found to be shifted to 1283–1289 cm−1, in the spectrum of 2, which indicates the coordination of this O atom. The low energy of the ν(C=O) vibration in the spectra of 1, that is, 1664 cm−1, is indicative that the carbonyl O-atom is involved in hydrogen bonding. The replacement of the hydrogen by the metal atom does not shift this band to lower frequency. Coordination of the imine nitrogen is also consistent with the presence of a band at 446 cm−1, assignable to ν(Sn–N). Bands at 284 and 248 cm−1 are assigned to ν(Sn–Oc=o) and ν(Sn–Oc-o), respectively [In the IR spectrum of Hrequency , 13, 16.ectively –21. 1H-NMR spectrum of 1, the N(3)-H resonance atδ12.30 and C–OH at δ 11.23 ppm indicates that these H atoms are involved in hydrogen bonding, 1H-NMR spectrum of 2, the formyl H-atom H–C(7) was shifted downfield upon coordination, which indicates variations in the electron density at position 7. Deshielding of carbons C4, C5, and C7 is observed in complexes, which is related to the electrophilicity of the tin atom. A σ-charge donation from the C–O and N donors to the tin center removes electron density from the ligand and produces this deshielding which will attenuate at positions remote from the tin center, In the 1 and 2 and the organotin oxide precursor of 2 were tested for their anti-proliferative activity in vitro against the cells of three human cancer cell lines: MCF-7 (human breast cancer cell line), T24 (bladder cancer cell line), A549 , and a mouse fibroblast L-929 cell line [see Pd(II) and Pt(II) complexes of 2-carbaldheyde thiosemicarbazone, HFoTsc, were found to be active in vivo against leukemia P388 cells and Pt(II) complexes of N4-ethyl 2-formyl and 2-acetylpyridine thiosemicarbazones showed cytotoxicity and were found to be able to overcome the cisplatin-resistance of A2780/Cp8 cells –16. Also50 values for H2HyFoSc (1) against the MCF-7 and A-549 cell lines are 164 and 175 μM, respectively, while against the L-929 and T-24 cell lines IC50 are >555 μM. Thus, 1 is considered as nonactive against these tested cell lines. Compound 2 is also considered nonactive against T24 cell line. The IC50 values for 2 against the L-929 and MCF-7 cell lines are 1.19 and 8.65 μM, respectively. These values are in the same range as observed for cisplatin and Ph2SnO, indicating that the observed cytotoxicity is probably due to the cytotoxicity of Ph2SnO. Compound 2 may then be considered as a vehicle for activation of the Ph2SnO as the cytotoxic agent. The IC50 value for 2 against A-549 is 0.086 μM and therefore 2 is significantly more active compared to Ph2SnO (47.1 μM) and cisplatin (1.53 μM), respectively. Compound 2 is thus 547.7 and 17.8 times more cytotoxic than the Ph2SnO and cisplatin, respectively, against this cell line. Thus, 2 exhibits selectivity and is considered as an agent with potential antitumor activity against A-549 tumor cell line and can therefore be a candidate for further stages of screening in vitro and/or in vivo.The IC


The neural cell adhesion molecule (NCAM) is highly expressed on the surface of small-cell-lung cancer (SCLC) cells. We have produced a monoclonal antibody, NY3D11, that binds to NCAM to investigate whether this antigen could be used to develop antibody-directed therapy for SCLC. 125I-labelled IgG and F(ab')2 fragments of NY3D11 localized selectively in human SCLC xenografts grown in nude mice. The human biodistribution of 131I-labelled NY3D11 after intravenous administration was investigated by gamma-camera imaging in six patients with SCLC. Three patients received IgG and three received F(ab')2. No evidence of localization to primary tumours or metastases was seen and antibody accumulated rapidly in the liver and bone marrow. The probable explanation for this distribution is that NY3D11 reacted with soluble NCAM or natural killer cells that possess the CD56 (NCAM) antigen.


High activity of cysteine proteases such as legumain and the cathepsins have been shown to facilitate growth and invasion of a variety of tumor types. In breast cancer, several recent studies have indicated that loss of the cysteine protease inhibitor cystatin E/M leads to increased growth and metastasis. Although cystatin E/M is normally expressed in the skin, its role in cysteine protease regulation and progression of malignant melanoma has not been studied.A panel of various non-melanoma and melanoma cell lines was used. Cystatin E/M and C were analyzed in cell media by immunoblotting and ELISA. Legumain, cathepsin B and L were analyzed in cell lysates by immunoblotting and their enzymatic activities were analyzed by peptide substrates. Two melanoma cell lines lacking detectable secretion of cystatin E/M were transfected with a cystatin E/M expression plasmid (pCST6), and migration and invasiveness were studied by a Matrigel invasion assay.Cystatin E/M was undetectable in media from all established melanoma cell lines examined, whereas strong immunobands were detected in two of five primary melanoma lines and in two of six lines derived from patients with metastatic disease. Among the four melanoma lines secreting cystatin E/M, the glycosylated form (17 kD) was predominant compared to the non-glycosylated form (14 kD). Legumain, cathepsin B and L were expressed and active in most of the cell lines, although at low levels in the melanomas expressing cystatin E/M. In the melanoma lines where cystatin E/M was secreted, cystatin C was generally absent or expressed at a very low level. When melanoma cells lacking secretion of cystatin E/M were transfected with pCST6, their intracellular legumain activity was significantly inhibited. In contrast, cathepsin B activity was not affected. Furthermore, invasion was suppressed in cystatin E/M over-expressing melanoma cell lines as measured by the transwell Matrigel assay.These results suggest that the level of cystatin E/M regulates legumain activity and hence the invasive potential of human melanoma cells. CST6 gene  thymidine (Amersham) for 24 h. Cells were detached with trypsin and 5 × 104 [3H]-labeled cells in 100 μl medium were added to each well in a 24-transwell permeable plate  coated with Matrigel basement membrane matrix . For migration studies the wells were left uncoated. The bottom wells received 600 μl medium with the same concentration of serum as the top well, either 1% or 10% FBS. Cells were removed from the upper and lower sides of the filter after 72 h incubation and [3H]-thymidine was counted in a liquid scintillation analyzer . The ratio of the counts obtained from the bottom versus total counts from both sides was used to determine migration and invasiveness.Cells were seeded at 2 × 10Legumain activity was measured by cleavage of the substrate Z-Ala-Ala-Asn-NHMec  as previously described ,45. CellInhibitory activity towards legumain was measured in the culture media from all the cell lines and from the pCST6-transfected cells (A375 and MCC11). Inhibitory activity was evaluated by measuring the residual enzyme activity in a preparation of legumain after the addition of conditioned media. As a source of legumain, homogenized rat kidney was applied to a cation exchange column  and eluted with a gradient of sodium chloride as described previously . Peak let-test, whereas two-way ANOVA was used to evaluate protease activities.Data are shown as the mean ± standard error of the mean (SEM). Statistical significance of the Matrigel invasion assays was evaluated using the two-tailed Student's Although it is known that cystatin E/M is secreted by keratinocytes and is present in the skin , little Another cysteine protease inhibitor, cystatin C, was detected as one band (15 kD) in media from most of the cell lines examined .The conditioned media from all cell lines was analyzed by fluorometry for their ability to inhibit the activity of a partially purified legumain fraction from rat kidney Fig. . LegumaiAlthough the activities of cathepsin B and L in melanoma and other tumor types are well documented, the expression and activity of legumain in melanoma is relatively unknown. To determine expression of legumain, cathepsin B and L, whole cell lysates were prepared from the same panel of cell lines as used for cystatin E/M analysis Fig.  followinBoth cathepsin B and L were detected in most of the cell lines, except those predominantly secreting the glycosylated form of cystatin E/M  was detected. When cells lacking cystatin E/M were transfected with an expression vector containing cystatin E/M cDNA (pCST6), a significant decrease in cellular legumain activity was observed, and conditioned media collected from the transfected cells had high inhibitory activity against exogenously added legumain. Furthermore, following transfection with pCST6, invasiveness was significantly decreased as measured in a Matrigel invasion assay. Collectively, our data strongly suggests that cystatin E/M inhibits melanoma progression by suppressing legumain activity.CST6 gene expression progressively decreases during disease progression [CST6 gene expression was observed. Furthermore, there was a significant decrease in CST6 expression between primary and metastatic melanoma when analyzed by microarray and quantitative RT-PCR. These data were the first to suggest that expression of CST6 decreases during progression to metastatic melanoma, a finding that is in agreement with previous studies in breast cancer [In a previous study comparing the gene expression profiles of primary and metastatic melanoma, it was shown that gression . When met cancer .CST6 functions as a tumor suppressor gene in melanoma, further studies are needed to determine the extent of cystatin E/M expression and the glycosylation status in cultured melanocytes and thin primary tumors.We have identified several cell lines derived from both primary and metastatic melanoma that secrete cystatin E/M. However, despite data suggesting that cystatin E/M has tumor suppressive capabilities, we did not detect lower levels in the metastatic versus the primary cell lines. This was probably due to the relatively small number of cell lines at our disposal originating from primary tumors, and furthermore, that most of these lines were derived from rather large lesions possibly having a phenotype more closely resembling metastatic rather than primary melanoma. Interestingly, the glycosylated 17 kD cystatin E/M form predominated in the melanoma samples, whereas the unglycosylated 14 kD form predominated in HaCaT keratinocytes. To confirm that in situ or primary melanoma.Although the expression of cystatin E/M in melanocytes has not been demonstrated, it has previously been shown that the protein is secreted from keratinocytes and thus present within the tumor microenvironment of the primary melanoma . Herein,By immunoblotting, we detected cystatin E/M secretion from four melanoma cell lines , and the conditioned media from these cells demonstrated the highest inhibitory activities against legumain. Surprisingly, we detected cystatin E/M by ELISA in media from only one of these melanoma cell lines. There are several potential explanations for this discrepancy, but the most likely being that there are fundamental differences between the applied methods. Immunoblotting measures denatured proteins while the ELISA attempts to detect the native cystatin E/M, possibly in complex with the interacting proteases. It is also possible that the amount of cystatin E/M in the media collected from the melanoma lines is below the limit of detection in the ELISA assay, as some cell lines showing strong expression on immunoblotting  had low amounts detected by ELISA. Furthermore, we utilized different antibodies in the two assays in this study and differences in the antibody affinity for the various forms of cystatin E/M are probable. The polyclonal antiserum used in the ELISA has, however, been demonstrated to recognize both the glycosylated and non-glycosylated forms of cystatin E/M (data not shown), making this explanation less likely. Nevertheless, there was a statistical positive correlation between the ELISA results for cystatin E/M and both molecular forms observed by immunoblotting.Some interesting relationships were observed between the different expression profiles of cystatin E/M, cystatin C, legumain, cathepsin B and L. First, in most melanoma cell lines examined, we identified an inverse correlation between secreted cystatin E/M and C. The only exception was the ocular melanoma cell line 92.1, in which neither of the cystatin isoforms was detected. Likewise, a similar inverse correlation between secreted cystatin E/M and both pro- and active forms of the examined proteases in lysates was observed. Legumain was expressed at very low levels in cell lines secreting cystatin E/M , while immunobands representing active legumain were high in all cells lacking the protease inhibitor. Similarly, with one exception (MCC70), there was no detectable expression of neither cathepsin B or L in the cells secreting cystatin E/M. Interestingly, this pattern was observed only in the melanomas and not in the cell lines derived from other cancer types. Although the expression levels of cystatin E/M and the proteases inversely correlated, an alternative explanation could be that cystatin E/M and protease expression levels are counter-regulated by some unknown mechanism. If so, the lower metastatic potential of the cystatin E/M expressing cells might be due to lower total expression levels of legumain, cathepsin B and L, respectively, and should be investigated further.Although legumain has been shown to impart metastatic properties in several different types of cancer, the present study is the first to address the expression and activity of the protease in melanoma. In general, high legumain expression and activity was revealed in the examined panel of melanoma cell lines. Two immunobands were repeatedly observed, one migrating at approximately 52 kD corresponding to the inactive proform of the enzyme, and the other at approximately 36 kD corresponding to the active form . The latTo determine the influence of cystatin E/M on protease activity and invasion, two cell lines (A375 and MCC11) with no basal secretion of cystatin E/M were transfected with pCST6. Cystatin E/M over-expression resulted in significantly increased ability to inhibit exogenously added legumain by the conditioned media, as well as decreased cellular legumain activity. The percentage decrease in legumain activity was greater in lysates from A375 than MCC11, possibly due to the lower basal level of legumain activity in A375. Surprisingly, cathepsin B was not affected by over-expression of cystatin E/M in these cells. Furthermore, a significant inhibition of invasive capacity was observed in both cell lines following transfection with pCST6. Since enhanced expression of cystatin E/M did not influence on the cell ability to migrate, the observed effect on invasion is most likely due to cystatin E/M-mediated inhibition of legumain activity. However, it is possible that the decreased invasiveness could in part result from effects on proteases other than legumain.in vitro motility and Matrigel invasiveness [in vitro growth whereas over-expression increased growth rate and tumor burden in vivo in pancreatic cancer [Although the majority of reports on cystatin E/M have indicated that it suppresses tumor growth and metastasis, there have been some conflicting data regarding the role of the protease inhibitor in cancer. It has been reported that cystatin E/M is up-regulated during the progression from primary to metastatic oropharyngeal squamous cell carcinoma . In addisiveness . A studyc cancer . The autc cancer -14.in vivo. The data presented provide new insight into the role of cystatin E/M in protease regulation and invasion in malignant melanoma.In summary, we have shown that cystatin E/M acts as a regulator of proteolytic activity of legumain and suppressor of invasion in metastatic melanoma. Further studies are needed to elucidate the role of both cystatin E/M and legumain in melanoma, including their expressions during different stages of the disease and their roles in tumor formation and metastasis Cys: cystatin; MCC: Moffitt Cancer Centre; pCST6: cystatin E/M (CST6)-encoding plasmidThe authors declare that they have no competing interests.JJB carried out cell culturing and harvesting, immunoblotting of cystatins, transfections, Matrigel invasion assays and statistical analysis, and participated in design of the study. MHH carried out immunoblotting and densitometry scanning of cystatins and proteases, participated in the statistical analysis and performed the final design of the figures. HTJ performed chromatography of rat legumain, participated in protease activity measurements and study design. AIR established several of the used melanoma cell lines and participated in the original conception and design of the study. MA performed ELISA measurements. ØF participated in the original design of the study. GMM participated in design and progression of the study. RS participated in protease activity measurements and statistical analysis, as well as in design and coordination of the study. JJB, GMM and RS drafted the manuscript, whereas all authors have contributed to, read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2407/10/17/prepub


Oncogene16: 933–934). When they were injected intravenously into athymic nude mice, KoTCC-1/BH formed more than three times as many tumour nodules in the lungs as did KoTCC-1/C. In addition, tumour progression, including lymph node metastasis and haemorrhagic ascites, was observed to be more advanced after the implantation of KoTCC-1/BH cells into the bladder wall of nude mice than after implantation of KoTCC-1/C cells. These enhanced malignant progression of KoTCC-1/BH cells were well correlated with anti-apoptotic activity under anchorage-independent conditions in in vitro experimental models. In contrast, there were no significant differences among these cell lines in their growth rates both in vitro and in vivo, invasive ability and cell motility. These findings suggest that, if it is overexpressed, Bcl-2 prolongs cell survival under unfavourable conditions encountered in the metastatic process, resulting in the enhanced metastatic potential of bladder cancer. © 1999 Cancer Research CampaignWe investigated the effect of Bcl-2 expression on the metastatic process of bladder cancer cells by using the Bcl-2-transfected human bladder cancer cell lines (KoTCC-1/BH) and the control vector only-transfected cell line (KoTCC-1/C), which were generated in our previous study


Histamine has recently been shown to be a growth factor for some gastric and colorectal cancer cells. Previous studies have shown that cimetidine blocks in vitro and in vivo histamine-stimulated growth and cAMP release from the human colonic cancer cell line, C170. In this study, ranitidine, another H2 receptor antagonist, did not affect either basal or histamine-stimulated in vitro proliferation of C170, and failed to prevent cAMP release in vitro. Ranitidine did not inhibit in vivo growth of C170 at a dose of 1, 10, 25, 50 or 100 mg/kg, in contrast to 50 mg/kg/day cimetidine, which produced 39.3% inhibition of tumour volume (p<0.01) after 23 days' treatment. Ranitidine did not inhibit in vivo histamine-stimulated growth of C170 cells . LIM2412, another colonic cancer cell line, was significantly stimulated by both cimetidine and ranitidine in vivo. Ranitidine had no effect on in vitro cell proliferation.


CDDP acts by formation of platinum (Pt)–DNA adducts; dFdC by dFdCTP incorporation into DNA, subsequently leading to inhibition of exonuclease and DNA repair. Previously, synergism between both compounds was found in several human and murine cancer cell lines when cells were treated with these drugs in a constant ratio. In the present study we used different combinations of both drugs (one drug at its IC25 and the other in a concentration range) in the human ovarian cancer cell line A2780, its CDDP-resistant variant ADDP, its dFdC-resistant variant AG6000 and two NSCLC cell lines, H322 (human) and Lewis lung (LL) (murine). Cells were exposed for 4, 24 and 72 h with a total culture time of 96 h, and possible synergism was evaluated by median drug effect analysis by calculating a combination index . With CDDP at its IC25, the average CIs calculated at the IC50, IC75 IC90 and IC95 after 4, 24 and 72 h of exposure were < 1 for all cell lines, indicating synergism, except for the CI after 4 h exposure in the LL cell line which showed an additive effect. With dFdC at its IC25, the CIs for the combination with CDDP after 24 h were < 1 in all cell lines, except for the Cls after 4 h exposure in the LL and H322 cell lines which showed an additive effect. At 72 h exposure all Cls were < 1. CDDP did not significantly affect dFdCTP accumulation in all cell lines. CDDP increased dFdC incorporation into both DNA and RNA of the A2780 cell lines 33- and 79-fold (P < 0.01) respectively, and tended to increase the dFdC incorporation into RNA in all cell lines. In the AG6000 and LL cell lines, CDDP and dFdC induced > 25% more DNA strand breaks (DSB) than each drug alone; however, in the other cell lines no effect, or even a decrease in DSB, was observed. dFdC increased the cellular Pt accumulation after 24 h incubation only in the ADDP cell line. However, dFdC did enhance the Pt–DNA adduct formation in the A2780, AG6000, ADDP and LL cell lines . This increase in Pt–DNA adduct formation seems to be related to the incorporation of dFdC into DNA (r = 0.91). No increase in DNA platination was found in the H322 cell line. dFdC only increased Pt–DNA adduct retention in the A2780 and LL cell lines, but decreased the Pt–DNA adduct retention in the AG6000 cell line. In conclusion, the synergism between dFdC and CDDP appears to be mainly due to an increase in Pt–DNA adduct formation possibly related to changes in DNA due to dFdC incorporation into DNA. © 1999 Cancer Research Campaign2′,2′-Difluorodeoxycytidine  and


ErbB-2 and EGFR  are expressed in lung adenocarcinomas and associated with a poor prognosis. Immunocytochemical analysis revealed erbB-2 and EGFR coexperession as a characteristic feature of most lung adenocarcinomas, and at levels of receptor expression present in bronchial epithelial cells. In primary lung tumours and cell lines, erbB-2 detected using Western blot analysis demonstrated low-level phosphotyrosine staining of the 185 kDa band, as compared with breast cancer cell lines. A549 and A427 lung adenocarcinoma cells treated with neu differentiation factor (NDF) showed increased erbB-2 phosphotyrosine staining, but to a much lesser extent than breast cancer cells. The lung cells were examined for expression of the potential autocrine growth factors NDF and transforming growth factor alpha  by Northern blot analysis. Both NDF and TFG-alpha mRNA were abundantly expressed in the A549 cells. NDF mRNA was highest during active cell proliferation and decreased in confluent cells or after treatment with the growth-inhibitory steroid dexamethasone. Primary tumours and cell lines expressed EGFR, showing higher basal level phosphotyrosine staining than erbB-2. Treatment with NDF and EGF  stimulated cell growth, and in A549 cells the presence of both factors provided an additive increase in cell growth. The growth stimulus that ligand-activated erbB-2 and EGFR provides to lung adenocarcinoma cells may establish a background of continued cell proliferation over which other critical transforming events may occur.


The cellular pharmacology of a series of C8-linked pyrrolobenzodiazepine dimers with polymethylene linkers of n = 3-6 (compounds 1-4) has been studied in a range of human tumour cell lines. The four compounds showed the same pattern of relative activity in five ovarian carcinoma cell lines and one cervical carcinoma cell line with the order of IC50 values of 1 < or = 3 < 4 < 2, which correlated with the previously demonstrated DNA interstrand cross-linking ability of the compounds in plasmid DNA. In human leukaemic K562 cells the agents produced a block in the G2/M phase of the cell cycle characteristic of cross-linking drugs, and extensive interstrand cross-linking was observed in cells by alkaline elution with no evidence of single-strand breaks. Cross-links continued to increase up to 24 h following a 1 h exposure to drug, and no repair was evident by 48 h. In a series of ovarian and cervical carcinoma cell lines with acquired resistance to cisplatin no cross-resistance to the most potent compound 1 was observed in two lines whose major mechanism of resistance to cisplatin was reduced platinum transport. Cross-resistance to 1 was observed in a cell line (A2780cisR) possessing elevated glutathione, and depletion of intracellular glutathione using D,L-buthionine-S,R-sulphoximine (BSO) from 10.25 nmol to 2.8 nmol 10(-6) cells reduced the level of resistance from 11-fold to 2-fold compared with sensitive cells. Cross-linking in the resistant cells was restored to 80% of the level in the parent line by BSO pretreatment. There was also a correlation between glutathione levels and sensitivity to 1 measured in several other ovarian cell lines. Compound 1 also showed cross-resistance in the doxorubicin-resistant cell line 41MdoxR and partial cross-resistance in CH1doxR cells. Both these lines possess elevated levels of p170 glycoprotein. Following treatment with 6 microM verapamil, the resistance in these lines decreased almost 2-fold and 8-fold respectively.


Taxol is a substrate for this transporter system and is used in the treatment of a wide range of human malignancies including lung, breast and ovarian cancer. We have generated a series of ovarian cell lines resistant to this compound, all of which overexpress MDR-1 through gene amplification. We present novel evidence that a constitutive activation of the ERK1/2 MAP kinase pathway was also observed although the level of active JNK and p38 remained unchanged. Inhibition of the ERK1/2 MAP kinase pathway using UO126 or PD098059 re-sensitised the Taxol resistant cells at least 20-fold. Importantly, when Mdr-1 cDNA was stably expressed in the wild-type cell line to generate a highly Taxol-resistant sub-line, 1847/MDR5, ERK1/2 MAP kinases again became activated. This result demonstrated that the increased activity of the signalling pathway in the Taxol-resistant lines was directly attributable to MDR-1 overexpression and was not due to the effects of Taxol itself. Additionally, we demonstrated that inhibition of the P13K pathway with LY294002 sensitised the MDR-1-expressing 1847/TX0.5 cells and 1847/MDR5 cells at least 10-fold but had no effect in the wild-type cells. This finding suggests a possible role for this pathway, also, in the generation of resistance to Taxol. © 2001 Cancer Research Campaign


The effect of sodium butyrate on mouse and human melanoma cell lines was evaluated. Sodium butyrate (0.1-2mM) is shown to reduce the clonogenic potential of several melanoma cell lines. The antiproliferative effect of sodium butyrate is accompanied by a marked increase in the activity of the plasma-membrane bound enzyme gamma-glutamyl transpeptidase. Sodium butyrate treated cells acquire a well developed rough endoplasmic reticulum and accumulate fat droplets. The development of the endoplasmic reticulum is associated with a marked increase in the activity of the enzyme marker NADPH cytochrome c reductase. It is suggested that the phenotypic alterations induced by sodium butyrate may serve as markers for the action of this agent on melanoma cells and other tumours.


The study of blocking factors requires in vitro assay of cell mediated immunity that parallels the in vivo response. By microcytotoxicity testing, progressor and immune peripheral blood lymphocytes caused significant target cell reduction. The cytotoxicity was specific as no cytotoxic effect was detected against unrelated normal as well as a malignant target cell lines. No anti-tumour effect was noted when progressor peripheral blood lymphocytes were evaluated in the Winn assay. In marked contrast, immune peripheral blood lymphocytes were capable of preventing tumour growth in the Winn assay. Furthermore, hamsters repeatedly immunized with irradiated SV40 tumour cells could resist a live cell challenge. Thus immune peripheral blood lymphocytes were chosen as the effector population to evaluate the abrogation ability of serum in the microcytotoxicity assay.


The main results of these studies can besummarized as follows: (1) exogenous or endogenous catecholamines suppress PBL functions through alpha-2-receptor-mediatedmechanisms, lymphocytes of the spleen are resistant to adrenergic in vivo stimulation,(2) direct or indirect cholinergic treatment leads to enhanced ex vivo functions of splenic and thymic lymphocytesleaving PBL unaffected, (3) cholinergic pathways play a critical role in the “talking back” of the immune system to the brain,(4) acetylcholine inhibits apoptosis of thymocytes possibly via direct effects on thymic epithelial cells, and maythereby influence T-cell maturation, (5) lymphocytes of the various immunological compartments were found to beequipped with the key enzymes for the synthesis of both acetylcholine and norepinephrine, and to secrete theseneurotransmitters in culture supernatantsFor several years, our group has been studying the


An in vitro osteolysis assay with 45Ca-labelled mouse calvaria has been used to investigate mechanisms of direct bone invasion by squamous carcinomas of the head and neck. Short-term (3-day) organ cultures of 8 fresh squamous carcinomas showed varying degrees of in vitro bone-resorbing activity which was blocked by indomethacin, an inhibitor of prostaglandin synthesis. Supernatant media from 6 established cell lines also induced bone resorption in vitro and evoked an osteoclastic response in the cultured calvaria. Osteolysis by supernatant media was not blocked by indomethacin in all the tumour-cell lines, and the production of non-prostaglandin osteolysins by the indomethacin-resistant lines is postulated. The two principal findings that emerge are: (1) Stimulants for osteoclastic activity are derived from both squamous-carcinoma cells and from host cells in the tumour stroma. (2) These stimulants are diverse. Indomethacin-sensitive agents, presumed to be prostaglandins, are most convincingly demonstrated in the fresh tumours. Indomethacin-resistant agents, presumably not prostaglandins, are more characteristic of the carcinoma cell lines.


The properties of a new tumour cell line (NB1-G) derived from human neuroblastoma by xenografting in nude rats followed by adaptation to tissue culture are described. Studies using a panel of monoclonal antibodies demonstrate the neuro-ectodermal nature of the cells and support the diagnosis of the primary tumour as neuroblastoma. Cytogenetic studies have revealed a human karyotype with several chromosomal abnormalities. Genetic analysis by in situ DNA hybridization has demonstrated the presence of multiple copies of the N-myc gene. Approximately 20-30 fold amplification of the gene is observed on Southern blot analysis. The cell line has been adapted to growth as multicellular tumour spheroids as well as monolayer culture. Radiobiological studies on spheroids show the cells to be radiosensitive with low capacity for sub-lethal damage accumulation and repair. The cell line should be useful for fundamental studies of human neuroblastoma as well as experimental therapy in vitro.


MDR1, MRP1 or MRP2 cDNA, and 2008 ovarian carcinoma cells stably expressing MRP1 cDNA, we have investigated in this study the specificity of the reversal agents V-104 (a pipecolinate derivative), GF120918 , and Pluronic L61 (a (poly)oxypropethylene and (poly)oxypropylene block copolymer). Transport experiments with cytotoxic drugs with polarized cell lines indicate that all three compounds efficiently inhibit MDR1 Pgp. Furthermore, V-104 partially inhibits daunorubicin transport by MRP1 but not vinblastine transport by MRP2. V-104 reverses etoposide resistance of 2008/MRP1 cells, whereas GF120918 does not reverse resistance due to MRP1. V-104 partially inhibits the export of the organic anion dinitrophenyl S -glutathione by MDCKII-MRP1 but not by MDCKII-MRP2 cells. Unexpectedly, export of the organic anion calcein by MDCKII-MRP1 and MDCKII-MRP2 cells is stimulated by Pluronic L61, probably because it relieves the block on entry of calcein AM into the cell by endogenous MDR1 Pgp. © 2000 Cancer Research CampaignThe human multidrug transporter MDR1 P-glycoprotein and the multidrug resistance proteins MRP1 and MRP2 transport a range of cytotoxic drugs, resulting in multidrug resistance in tumour cells. To overcome this form of drug resistance in patients, several inhibitors  of these transporters have been isolated. Using polarized cell lines stably expressing human


Sir,British Journal of Cancer (2007) 97: 934–940, we indeed did not check the authenticity of the cell lines MCF-7/ADR and KB, so, it is likely that MCF-7/ADR is not an authentic variant of the MCF-7 breast carcinoma cell line and that KB is not derived from oral carcinoma, although there are numerous papers to mention that MCF-7/ADR is a variant of the MCF-7 breast carcinoma cell line and KB is derived from oral carcinoma. However, the authenticity of these cell lines does not compromise the conclusion and scientific significance of this paper. In our article, it is very clear that these cell lines (MCF-7/ADR and KBV200) are drug resistant and their drug resistances are mediated by P-glycoprotein. These characteristics of these cell lines are enough to justify them to be used to study the pharmacologic effects of ZD6474 and the mechanisms involved.In the article ZD6474 reverses multidrug resistance by directly inhibiting the function of P-glycoprotein in


We have produced adriamycin (ADM)-resistant variants of the human lung cancer cell lines NCI-H69 , MOR (adenocarcinoma) and COR-L23 (large cell) but have failed to produce resistant variants of two other small cell lines. In each case, the derivation protocol took 7-9 months and included a period of drug-free growth. All three resistant lines show reduced cellular content of ADM after 1 h exposure when compared with their controls. During prolonged incubation of control and resistant NCI-H69 cells in 0.4 microgram ml-1 ADM, the ADM content of resistant cells was 6-7 times lower than that of control cells. The ratio of ADM doses to suppress growth of the two lines, however, was in the range of 40-200X. The ADM-resistant variant of NCI-H69 was also resistant to vincristine, colchicine, VP16, mitozantrone, 4' epiadriamycin and 4' deoxyadriamycin, somewhat resistant to melphalan but not resistant to aclacinomycin A, bleomycin of CCNU. The resistance to ADM could be partially overcome by the use of verapamil, an inhibitor of calcium transport.