Treatment of non-small cell lung cancer (NSCLC) remains a difficult task in oncology. Targeted inhibition of oncogenic proteins is promising. In this study, we evaluate the expression of MET and PKCß and in vitro effects of their inhibition using SU11274 and enzastaurin (LY317615.HCl) respectively.Patient samples were analyzed by immunohistochemistry for expression of PKCß and MET, utilizing tissue microarrays under an IRB-approved protocol. Expression of PKCß and MET was evaluated in cell lines by immunoblotting. Treatment with SU1174 against MET and enzastaurin against PKCß was performed in H1993 and H358 cell lines, and cell proliferation and downstream signaling  were evaluated by immunoblotting. Statistical analysis was performed using SPSS 16.0.Expression of MET positively correlated with lymph node metastases (p=.0004), whereas PKCß showed no correlation (p=0.204). MET and PKCß expression were also strongly correlated (p<0.001). Expression of MET was observed in 5/8 cell lines , whereas PKCß expression was observed in 8/8 cell lines. Cell proliferation was significantly impaired by treatment with SU11274 and enzastaurin, and their effects were synergistic in combination (CI=0.32 and 0.09). Phosphorylation of MET, FAK, AKT, and GSK3ß were strongly inhibited with both agents in combination.Concomitant inhibition of MET and PKCß significantly increased cytotoxicity in vitro against NSCLC, disrupting important downstream signaling pathways. Further evaluation in animal models is warranted. Lung cancer is the most common cause of cancer mortality in the United States and worldwide.2 The disThe protein kinase C (PKC) family of serine-threonine protein kinases has been implicated in several important cellular functions including proliferation, motility, invasion, and apoptosis. Of the v12The c-MET receptor tyrosine kinase (MET) was originally identified as the cellular homologue of the TPR-MET oncoprotein. MET is oin vitro. Intracellular signaling networks were also evaluated for effects of the combination therapies.Combination therapies, especially those involving MET inhibition, have been increasingly studied as a treatment strategy in lung cancer.–24 GivenParaffin-embedded, formalin-fixed tumor tissues from patients with NSCLC with associated clinical information  were processed into a tissue microarray (TMA) under an Institutional Review Board-approved protocol (UCMC #9571 and #13473). Immunohistochemistry (IHC) was performed using biotin-free HRP-labeled polymer complex bound to secondary antibody , and performed according to previously published procedures. NegativeNCI-H522 (adenocarcinoma), NCI-H1703 (adenocarcinoma), A549 (adenosquamous carcinoma), NCI-H1993 (adenocarcinoma), NCI-H2170 (squamous cell carcinoma), NCI-H661 (large cell carcinoma), SW1573  and NCI-H358  cell lines were obtained from the American Type Culture Collection  All cell lines were cultured as per our established protocols.Enzastaurin was provided by Eli Lilly . Fetal bovine serum (FBS) was obtained from Gemini Bioproducts . SU11274 was obtained from EMD Biosciences . Cell culture media, penicillin, and streptomycin were obtained from Cellgro . Antibodies used included: anti-PKCß2, anti-Met, anti-phospho-FAK (Tyr925), anti-FAK, anti-phospho-AKT (Ser473) and anti-phospho-GSK3ß (Ser9) ; anti-phospho-Met (Tyr130/1234/1235) ; ß-actin monoclonal antibody . All other chemicals were purchased from Sigma .μM enzastaurin, 2.5 μM SU11274, or enzastaurin and SU11274 in combination (or DMSO as a negative control) for different durations as described, in serum-free media. Whole cell lysates were collected and immunoblotting was performed following routine protocols.[To examine protein expression in NSCLC cell lines under basal conditions, subconfluent cells were cultured in medium supplemented with 10% FBS. To detect the inhibition of cell transduction pathways, cells grown on 10-cm culture dishes for 24 hours were washed twice with PBS and incubated at 37°C with 2.5 rotocols. The sameCells were plated in 96-well plates at 5×10^3 cells per well in serum-containing media and grown for 24 hours. Drugs (or drug carrier) were added in serum-free media, and cells were incubated for 72 hours. Cell growth was estimated utilizing fluorometric readings after the addition of Alamar Blue , a non-radioactive, non-toxic dye that is reduced and whose fluorescence is proportional to cellular metabolic activity. A HT Synergy Plus microplate reader  was used to measure fluorescence (530-560 nm excitation wavelength and 590 nm emission). Drug synergism was estimated by the median-effect analysis using thComparisons between average means of protein expression were made by utilizing the Student's t-test for independent samples. For evaluation of differences in frequencies among different categories, the Chi-square test was used. The statistical software used was SPSS, version 16.0 .Evaluation of protein expression of PKCß and MET in the TMAs was performed. In order to further investigate the potential therapeutic implications of concomitant PKCß and MET inhibition, eight NSCLC cell lines were evaluated for protein expression by immunoblotting. MET expression was robustly observed in 5/8 cell lines , while PKCß expression was seen in all 8 cell lines .Cell growth inhibition with various drugs was evaluated in H1993 and H358 cell lines. After treating these cells for 72 hours with SU11274, enzastaurin, or both drugs in combination in increasing concentrations, it was observed that the combination was synergistic in causing significant effects on cell proliferation in both cell lines. Combinatorial indices for H358 cells at ED50 was 0.32, and for H1993 was 0.09, therefore proving significant synergistic effect .In order to investigate the molecular effects of treatment with SU11274 and/or enzastaurin, immunoblotting with phospho-specific antibodies was carried out in H1993 cells treated with these drugs for the time periods shown . TreatmeThere have been multiple examples of the utilization of a combination of targeted agents in lung cancer. Unfortunately, the accumulated experience has been largely unsuccessful in the treatment of this disease. Our work presents a combination that might prove to be biologically relevant, and perhaps have a reasonable toxicity profile to be investigated in the clinical setting. Herein, we have identified that MET and PKCß are expressed in lung cancer, and their inhibition can be synergistic. Interestingly, also, metastasis is common in lung cancer, and lymph node metastasis correlated with both MET and PKCß expression.For nearly two decades, MET has been known to be commonly aberrant in lung cancer, whether overexpressed,2829 ampl2834MET activation can lead to autophosphorylation or phosphorylation of downstream intermediates and activation of signaling pathways. As an example, in small cell lung cancer (SCLC), activation of MET with HGF leads to phosphorylation/activation of several pathways involving cell proliferation/survival , cell cycle (RB), and cytoskeletal proteins .in vitro and in vivo settings.[MET has also been shown to signal synergistically with EGFR as part of a broad signaling network that cooperatively drives activation of these downstream pathways.40 Indeedsettings.in vitro effect of enzastaurin against malignant pleural mesothelioma,[in vitro activity of enzastaurin and pemetrexed, a commonly used antifolate compound, has also revealed synergistic activity against SW1573 and A549 cell lines. Multiple biochemical pathways were shown to be affected, such as cell cycle control, apoptosis, and angiogenesis.[Inhibition of PKCß with enzastaurin has been recently studied in thoracic malignancies. Our previous work has demonstrated the thelioma, and its ogenesis. In anothogenesis. The effiogenesis. A phase ogenesis.in vitro assays. This finding was associated with decrease in activation of downstream effectors such as GSK3ß, AKT and FAK. These data suggest that concomitant inhibition of MET and PKCß may be an effective treatment strategy for NSCLC, especially for those patients whose tumors have developed prior tyrosine kinase resistance. These findings warrant further investigation in vivo to determine whether such a dual inhibition strategy is effective in reducing tumor progression.We have shown here that MET and PKCß tend to be coexpressed in NSCLC cell lines and tissues and that simultaneous inhibition of MET and PKCß significantly decreased cell proliferation in


Retinoids and vitamin D are important factors that regulate cellular growth and differentiation. An additive growth-inhibitory effect of retinoids and vitamin D analogues has been demonstrated for human myeloma, leukaemic and breast cancer cells. We set out to study the effects of the vitamin D analogue EB1089 and the retinoids all-trans- and 9-cis-retinoic acid on the human pancreatic adenocarcinoma cell lines Capan 1 and Capan 2 and the undifferentiated pancreatic carcinoma cell line Hs766T. The cell lines investigated expressed vitamin D receptor, retinoic acid receptor (RAR)-alpha and gamma as determined by polymerase chain reaction after reverse transcription. RAR-beta was expressed only in Hs766T cells. Addition of all-trans-retinoic acid increased the amount of RAR-alpha mRNA in the three cell lines and induced RAR-beta mRNA in Capan 1 and Capan 2 cells. All-trans-retinoic acid at a concentration of 10 nM inhibited the growth of Capan 1 and Capan 2 cells by 40% relative to controls. 9-cis-Retinoic acid was less effective. Neither all-trans-retinoic acid nor 9-cis-retinoic acid affected the growth of Hs766T cells. EB1089, if added alone to the cells, did not significantly inhibit growth. However, the combination of 1 nM EB1089 with 10 nM all-trans-retinoic acid exerted a growth-inhibitory effect of 90% in Capan 1 cells and of 70% in Capan 2 cells. Our data suggest that vitamin D analogues together with retinoids inhibit the growth of human pancreatic cancer cells. However, in vivo studies are necessary to examine the potential use of retinoids and vitamin D analogues on pancreatic cancer.


Verapamil, the prototype calcium channel blocker, reversibly inhibits cell proliferation in many normal and tumour cell lines . We have found that two closely related cell lines - B16 murine melanoma cells and B10.BR normal murine melanocytes growing in culture - behave differently in the presence of verapamil, and we are now utilising these two related cell lines to help elucidate the molecular basis of verapamil's antiproliferative effect. In this study, we studied cell cycle phase distribution and c-myc gene expression in both cell lines in the absence of verapamil, during incubation with verapamil and after the cells were washed free of verapamil. Our studies show that 100 microM verapamil rapidly blocks DNA synthesis in melanocytes but not in B16 cells. Similarly, incubation with verapamil for 6-24 h results in a decreased c-myc signal in melanocytes, but a transient increase in c-myc expression in B16 cells. After verapamil is washed from the cells following a 24-h incubation with drug, c-myc expression increases in melanocytes as they begin again to proliferate, but decreases in B16 cells as they begin to die. Our disparate results with these cell lines suggest that c-myc gene expression, regardless of its known involvement in growth control, is not the immediate target for verapamil's inhibitory action.


This study investigates the response to pemetrexed in a panel of eight mesothelioma cell lines and the clinical outcome for patients treated with pemetrexed in relation to folate receptor alpha (FR50). FRα expression was determined by western blotting and that of FRα, reduced folate carrier (RFC) and proton-coupled folate transporter (PCFT) by real-time quantitative RT–PCR. Immunohistochemistry for FRα was carried out on 62 paraffin-embedded samples of mesothelioma from patients who were subsequently treated with pemetrexed.Cell lines were treated with pemetrexed to determine the concentration that reduced growth to 50%  and RS5 cells having a GI50 value greater than 10 μM. No FRα protein was detected in any cell line, and there was no relationship between sensitivity and expression of folate transporters. FRα was detected in 39% of tumour samples, generally in a small percentage of cells. There was no correlation between the presence of FRα and the outcome of pemetrexed treatment, and no significant difference between histological subtypes.A wide range of GIα.Response to treatment with pemetrexed does not depend on the presence of FR Malignant pleural mesothelioma (MPM) is an aggressive tumour of the pleura and usually has a poor prognosis. It is increasing in many countries worldwide as a result of widespread exposure to asbestos in the past and is expected to peak around 2015 in the United Kingdom . Inhibition of TS leads to depleted levels of thymidine, which are crucial to DNA synthesis. Pemetrexed also inhibits, to a lesser degree, glycinamide ribonucleotide formyltransferase (GARFT) (α) is an energy-dependent high-affinity, low-capacity folate-binding protein that delivers folates and antifolates into cells. The low capacity is due to endocytosis of the receptor that has to return to the cell surface in order to function. Expression is limited to specific tissues and is expressed in a characteristic distribution, for example, on syncytiotrophoblastic cells of the placenta and the proximal convoluted tubule of the kidney  and was 14-fold more sensitive when maintained in sub-physiological levels (1 nM leucovorin) (Folic acid and antifolates enter cells by three mechanisms. The reduced folate carrier (RFC) is a bidirectional anion exchanger that has a high affinity for reduced folate co-factors and antifolates, but a low affinity for folic acid. It has a neutral pH optimum and is ubiquitously expressed ( cancers . Howevercovorin) . Membranα to pemetrexed transport is likely to be significant in cells in which FRα is highly expressed. In these situations, transport of pemetrexed via FRα may enhance delivery of the drug to the tumour and potentially enhance response. Recently, a new monoclonal FRα antibody has been developed that can be used for immunohistochemistry on formalin-fixed paraffin-embedded (FFPE) tissues  (Sigma-Aldrich) or 10% dialysed FBS (DFBS) . Dialysis of serum removes low molecular weight compounds such as thymidine and homocysteine, which may affect the response of cells to pemetrexed. The concentration of folic acid in the medium used is 2 μM. All cell lines were subcultured regularly and were negative for mycoplasma.Eight human mesothelioma cell lines were used in this study. Five cell lines were of epithelioid type: NCI-H28 (H28), NCI-H2052 (2052), NCI-H2452 (2452) , NCI-H226 (H226)  and JL1 ; MSTO-211H (MSTO) (ATCC) was of biphasic origin; two cell lines, DM3 and RS5 (DSMZ) were of sarcomatoid type. Cells were grown in RPMI 1640  containing 2 m3 cells per well and treated with 10 nM to 10 μM pemetrexed for approximately three cell doubling times, apart from the slower growing RS5 and DM3 cells that were treated for 6 days. The concentration of pemetrexed resulting in 50% growth inhibition (GI50) for each cell line was calculated using GraphPad Prism software.Growth-inhibition studies using pemetrexed were carried out using the SulfurRhodamine B (SRB) .Cell lysates required for western blotting were prepared after washing cells with ice-cold PBS and addition of lysis buffer  from each sample were loaded on a 4–20% gradient polyacrylamide gel (Invitrogen). After SDS–PAGE and electroblotting , as well as universal mastermix. The expected amplicon sizes were 89, 106, 72 and 187, respectively. PCR was performed as follows: 50°C for 2 min, 95°C for 10 min, and 40 cycles of 95°C for 15 s and 60°C for 1 min using the ABI Prism 7900HT sequence detection system (Applied Biosystems). Results were quantified relative to 18S, and calibrated to the IGROV1 FRα estimation.RNA was extracted from cells using the Qiagen RNAeasy minikit  and reverse transcribed using TaqMan reverse transcriptase reagents . A volume of 2.5 −2, i.v. administered over 3 h on day 1) and pemetrexed administered at a dose of 500 mg m−2 i.v. for 10 min on day 1. Cycles were repeated every 21 days to a maximum of six cycles. Patients also received folic acid supplementation at a dose of 400 μg daily, beginning at least 7 days before the first dose of pemetrexed and one injection of 1000 μg vitamin B12 intramuscularly 7–14 days before beginning the treatment. Folic acid was continued, and vitamin B12 was repeated every 12 weeks, throughout the treatment until 21 days after the last dose of pemetrexed. Dexamethasone, 4 mg twice daily, was given orally on the day before, the day of and the day after therapy. Our antiemetic regimen included a serotonin antagonist. Chemotherapy was discontinued in the event of disease progression, unacceptable toxicity or after completion of six cycles. Cycles were repeated at 100% of the intended doses if neutrophil and platelet counts were ⩾1.5 × 109 l−1 and ⩾100 × 109 l−1, respectively. If any dose reduction occurred, patients continued to receive the reduced dose throughout their treatment. All patients underwent planned radiographic evaluation.Eligible patients were identified from chemotherapy prescription records. All patients underwent histological diagnosis of malignant pleural mesothelioma and were treated according to our institutional protocol, in which carboplatin was administered to produce a value of the area under the curve (AUC) of 5, i.v. over 30 min on day 1  and allowed to dry at 56°C. The slides were dewaxed in xylene, rehydrated in graded ethanol and incubated for 10 min in 0.5% hydrogen peroxide solution to block endogenous peroxidase activity. The slides were then treated with an endogenous biotin blocking kit (Dako) before high-temperature antigen retrieval (microwaved thrice for 5 min in citrate buffer pH 6.0). Blocking with a 1 in 5 dilution of normal goat serum for 10 min preceded overnight incubation in a 1 in 20 dilution of FRα antibody (Sections of 4 Staining was graded independently by two assessors (JN and AR) using a composite index. The intensity of staining was scored 0–3, with 0 being no staining, 1 weak staining, 2 moderate staining and 3 intense staining. This was multiplied by the percentage of tumour area stained, using 10 for upto 10%, 25 for 11–25%, 50 for 26–50%, 75 for 51–75% and 100 for 76–100% of the tumour area stained, giving a composite score range of 0–300. The staining obtained in the renal tissue was used as a reference for intense staining, with a score of 300.α immunohistochemistry of tumour samples was correlated to clinical parameters such as response (objective decrease in tumour size on CT imaging), disease control rate , time-to-treatment failure  and overall survival .The composite score for the FRThis study was approved by the local research ethics committee and the Newcastle Hospitals Caldicott Guardian.50±s.d. value from three replicate experiments is shown in 50 values ranged from 14 nM in the H2452 epithelioid cell line to greater than 10 μM in the sarcomatoid cell line RS5. In most cases, apart from sarcomatoid RS5 cells, those grown in medium containing DFBS were more sensitive to pemetrexed than cells grown in medium containing normal FBS, with the largest difference observed in JL1 and DM3 cell lines. This is probably because of the removal of molecules such as folates, thymidine and homocysteine from the serum on dialysis, which are able to rescue cells from the effects of pemetrexed.Pemetrexed inhibited the growth of mesothelioma cell lines to variable extents. Representative graphs showing patterns of growth inhibition for each cell line in medium containing FBS or DFBS are shown in α protein in any of the mesothelioma cell lines, but was shown to be present at high levels in the IGROV1 ovarian cell line positive control. Similarly, the results from real-time PCR showed FRα mRNA to be undetectable in three cell lines , whereas extremely low levels at the limit of detection were present in the other mesothelioma cell lines. In these cell lines, the level of expression for FRα was approximately 100 times less than the highly expressing IGROV1 ovarian cancer cell line.The results for western blotting and quantitative RT–PCR are shown in 50 of pemetrexed in the cell lines.The results from real-time RT–PCR for other folate transporters, RFC and PCFT, in the cell lines are shown in In all, 62 patients were eligible for this study. A summary of demographics and treatment details is shown in α expression was considered to be positive when the composite score was >0. FRα expression was positive in 24 of the 62 samples examined . Both membranous and cytoplasmic staining was seen. When present, however, FRα expression was usually weak as 12 (50%) stained samples had a score of 30 or less. Only two samples had a composite score of more than 100. FRα expression was seen to be higher in the epithelioid disease subtype but was not significant , biphasic, n=6 (35%) and sarcomatoid, n=2 (22%), × 2 1.614, P=0.45).A sample of the positive control and various staining intensities are depicted in α. Responses were seen in 8 (33%) and 9 (24%) patients with positive and negative FRα-stained tumours , whereas 75 and 61%, respectively, had disease control . The TTF and OS rates were not statistically different between patients with positive FRα tumours and those that were negative , as shown in α.There were no significant differences in the proportion of patients with a response or disease control when subdivided into those with a positive or negative expression of FRα in the uptake of both folates  and antifolates has been the subject of numerous publications with contrasting results  were less sensitive to pemetrexed than the other cell lines. Clinically, sarcomatoid tumours tend to be more aggressive than epithelioid tumours . It has been reported that the effect of pemetrexed is enhanced in cell lines when there is a low or physiological concentration of folate in the medium  in mesothelioma of differing histological type. Following the development of an FRα antibody for use on paraffin-embedded tissues , involved in α expression in mesothelioma, or to the expression of RFC and PCFT in the cell lines. Other reasons for differences in sensitivity to pemetrexed remain to be established, but this study shows that there is no simple relationship between the sensitivity of cell lines to pemetrexed and the expression of any of the folate transporters; other potential molecular markers for pemetrexed response in mesothelioma are currently under investigation.In conclusion, the use of a varied panel of eight mesothelioma cell lines and the investigation of a large number of tumour samples from patients with mesothelioma treated with pemetrexed in this study demonstrate that there is no relationship between response to pemetrexed and FR


Cooperative synergistic effects by these two ligands have been reported, and it is, therefore, possible that greater therapeutic effects could be achieved if these compounds were administered together. The role of retinoid-dependent anti-activator protein 1 (anti-AP-1) effects in controlling cancer cell proliferation appears significant. We have utilized an anti- AP-1 retinoid , which does not transactivate through a retinoic acid response element (RARE), and a potent vitamin D3analogue  together at low, physiologically safer doses against a panel of prostate cancer cell lines that represent progressively more transformed phenotypes. The LNCaP (least transformed) and PC-3 (intermediately transformed) cell lines were synergistically inhibited in their clonal growth by the combination of LH and SR11238, whereas SR11238 alone was essentially inactive. DU-145 cells (most transformed) were completely insensitive to these analogues. LNCaP cells, but neither PC-3 nor DU-145, underwent apoptosis in the presence of LH and SR11238. Transactivation of the human osteocalcin vitamin D response element (VDRE) by LH was not enhanced in the presence of SR11238, although the expression of E-cadherin in these cells was additively up-regulated in the presence of both compounds. These data suggest the anti-AP-1 retinoid and the vitamin D3 analogue may naturally act synergistically to control cell proliferation, a process that is interrupted during transformation, and that this combination may form the basis for treatment of some androgen-independent prostate cancer. © 1999 Cancer Research CampaignThe secosteroid hormones, all-


Decreased accumulation of the fluorescent dye BCECF  characterized murine and human multidrug-resistant cell lines overexpressing the multidrug resistance protein (MRP). Indomethacin (10 microM), a known cyclo-oxygenase and glutathione-S-transferase inhibitor as well as a modulator of anion transport, increased accumulation and blocked efflux of BCECF in MRP-expressing murine and human cells. The drug did not affect P-glycoprotein (P-gp)-mediated export of rhodamine 123. The indomethacin effect on BCECF efflux was not reversed by the addition of exogenous prostaglandins, suggesting that the drug acts by a mechanism other than decreasing prostaglandin synthesis. Indomethacin also increased multidrug susceptibility of both murine and human cell lines overexpressing MRP, but not those displaying P-gp-associated resistance. In addition, indomethacin modulated the decreased vincristine accumulation in cells expressing MRP, but not in those expressing P-gp. These data suggest that indomethacin is a specific inhibitor of MRP, possibly functioning by inhibition of glutathione-S-transferase or, alternatively, by direct competition with the drug at the transport site.


Attempts have been made by a number of methods to eliminate minimal residual disease from bone marrow to be reinfused in autologous transplantation. In this paper we describe a conjugate containing a monoclonal antibody, named 8A, recognising a plasma cell-associated antigen, and momordin, a ribosome-inactivating protein similar to the ricin A-chain. This immunotoxin is active on target cell lines and on neoplastic plasma cells, while myeloid progenitors are fairly resistant. The conjugate is shown to be acceptable for ex vivo purging in autologous bone marrow transplantation in multiple myeloma patients.


Thromboplastic and fibrinolytic activities of 14 lines of cultured human cancer cells were estimated by modified Astrup's methods. High tissue thromboplastic activity was found in one line of urinary-bladder cancer, 2 lines of gastric cancer and one line of lung cancer, but no activity was found in 6 lines of lung cancer. High fibrinolytic activity was noted in one line of gastric cancer and 2 lines of lung cancer, but no activity was seen in 6 lines of lung cancer and one line of gastric cancer. No plasmin activity was found. The tumour cell lines could be classified into 3 groups on the basis of the 2 activities. Cancer cell lines could also be classified into 2 groups: with high or low release of thromboplastin into culture media. Fibrinolytic activity was found in the culture media of all cell lines with high fibrinolytic activity. Fibrinolytic activity, but not thromboplastic activity, seemed to be influenced by the constituents of culture media. No definite correlation was found between the 2 activities and the histological types of the parent tumours of the cultured cells.


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.