Casearia sylvestris , popularly known as “guaçatonga”, is a plant widely used in folk medicine to treat various diseases, including cancer. The present work deals with the chemical composition as well as the cytotoxic evaluation of its essential oil, its main constituent and derivatives. Thus, the crude essential oil from leaves of C. sylvestris was obtained using a Clevenger type apparatus and analyzed by GC/MS. This analysis afforded the identification of 23 substances, 13 of which corresponded to 98.73% of the total oil composition, with sesquiterpene α-zingiberene accounting for 50% of the oil. The essential oil was evaluated for cytotoxic activity against several tumor cell lines, giving IC50 values ranging from 12 to 153 μg/mL. Pure α-zingiberene, isolated from essential oil, was also evaluated against the tumor cell lines showing activity for HeLa, U-87, Siha and HL60 cell lines, but with IC50 values higher than those determined for the crude essential oil. Aiming to evaluate the effect of the double bonds of α-zingiberene on the cytotoxic activity, partially hydrogenated α-zingiberene (PHZ) and fully hydrogenated α-zingiberene (THZ) derivatives were obtained. For the partially hydrogenated derivative only cytotoxic activity to the B16F10-Nex2 cell line (IC50 65μg/mL) was detected, while totally hydrogenated derivative showed cytotoxic activity for almost all cell lines, with B16F10-Nex2 and MCF-7 as exceptions and with IC50 values ranging from 34 to 65 μg/mL. These results indicate that cytotoxic activity is related with the state of oxidation of compound. Casearia was traditionally regarded as belonging to the family Flacourtiaceae. However phylogenetic and chemical studies, besides ecological and morphological observations showed that Flacoutiaceae is, in fact, composed by two clades, with the genus Casearia belonging to the Salicaceae + cation peak at m/z 123. A further fragmentation of this peak with loss of ethylene produced the base peak of spectrum m/z 95 , suggesting that this sesquiterpene may be primarily responsible for the cytotoxic activity against this particular cell line.The crude oil displayed activity against all cell lines tested with IC50 of 65.2 μg/mL, and IC50 values greater than 200 μg/mL for the other tumor cell lines, indicating that PHZ has low cytotoxic activity against these tumor cells.Additionally, it was observed that at the tested concentrations PHZ was only active against the B16F10-Nex2 cell line, with an IC50 values ranging from 34–65 μg/mL. The higher lipophilicity of totally hydrogenated zingiberene, compared to other compounds, could be responsible for enhanced cell penetration through the plasma membrane. The totally hydrogenated zingiberene (THZ) showed cytotoxic activity against almost cell lines, except for B16F10-Nex2 and MCF-7, with ICC. sylvestris, Salvador et al. [C. lasiophylla were cytotoxic against tumor cell lines U251, UACC-62, MCF-7, NC1-ADR/RES, NCI-H460, PC03, OVCAR-3, HT-29 K562. Silva et al. [C. sylvestris collected in Campinas against several tumor cell lines . However, these oils were shown to be composed by bicyclogermacrene and β-caryophyllene as main compounds, a different profile of that detected in the present work.Although there are no studies concernng the cytotoxic activity of sesquiterpenes isolated from oil of r et al.  showed ta et al. , also shCasearia sylvestris with no deformation and degradation by exogenous factors were selected and collected with the aid of sterile pliers from a single tree at the Atlantic Forest area in São Paulo city, SP, Brazil , in October, 2012.Leaves of C. sylvestris were extracted over 5 h by steam distillation in a Clevenger type apparatus to afford 2.9 g of crude essential oil. The oil was analyzed by GC-FID and GC-LR-EI-MS using a RtX-5 capillary column. The identification of the individual compounds was performed by comparison of retention indexes  and comparison of recorded mass spectra with those available in the system library [2Cl2 in a split mode (1:10) employing helium as the carrier gas (1 mL/min) under the following conditions: injector and detector temperatures of 220 °C and 250 °C, respectively; oven programmed temperature from 40–240 °C at 3 °C/min, holding 5 min at 240 °C. GC-FID was performed in quantitative analysis. The percentage compositions of the oil samples were computed by internal normalization from the GC peak areas without using correction for response factors. GC-LR-EI-MS analysis was conducted on a Shimadzu GC-17A chromatograph interfaced with a MS-QP-5050A mass spectrometer. Helium was used as the carrier gas. The LR-EI-MS operating conditions were an ionization voltage of 70 eV and an ion source temperature of 230 °C with the same conditions described above.Fresh leaves  of  library . GC chro2 gel soaked with AgNO3 (10%) column (63 cm × 5 cm i.d.) chromatography eluted with CH2Cl2-acetone in proportions of 99:1, 95:5 and 90:10 (200 mL for each eluent) to afford 30 fractions which were individually analyzed using GC-FID and then pooled into nine groups (A to I). Fraction F was composed of pure α-zingiberene (203 mg).Part of crude oil (2.0 g) was subjected to flash chromatography on SiOα-Zingiberene. 1H-NMR : 2.01 m , 5.38 brs , 5.57 dd , 6.70 dd , 2.14 m , 1.16 m , 1.48 m , 1.99 m , 5.02 brt , 1.61 s , 1.64 s , 0.83 d , 1.68 d . 13C-NMR : 26.2 (C-1), 120.6 (C-2), 131.6 (C-3), 128.1 (C-4), 131.3 (C-5), 38.3 (C-6), 34.5 (C-7), 36.3 (C-8), 24.7 (C-9), 125.0 (C-10), 131.4 (C-11), 26.2 (C-12), 17.9 (C-13), 17.8 (C-14), 21.3 (C-15). LR-EI-MS (70 eV): m/z (rel. int.): 204 (9), 145 (26), 132 (90), 119 (98), 105 (62), 91 (43), 55 (47).2/g) and hexane (5 mL) were added. After addition of H2 (8 atm), the mixture were stirred for 3 h at 90 °C. Then, the reactor was cooled and the product was filtered [1H-NMR : 1.60 m , 2.01 m , 5.32 m , 2.04 m , 1.63 m , 1.16 m , 1.25 m , 1.23 m , 1.62 m , 0.91 d , 0.86 d , 1.66 s . This data suggests that the remaining double bond is located as shown in 2, Ni-Raney 20%, 30 mg, 120 °C, 3 h) to obtain 60 mg (65%) of the fully saturated product THZ. In a high-pressure reactor (stainless steel), pure α-zingiberene , Ni-Raney catalyst . The melanotic B16F10-Nex2 subline, characterized at the Experimental Oncology Unit , is characterized by low immunogenicity and moderate virulence. Human melanoma (A2058), glioblastome (U87), leukemia (HL-60), uterus carcinoma (Siha) and breast cancer (MCF-7) cell lines were obtained from the Ludwig Institute for Cancer Research. Human cervical carcinoma (HeLa) was acquired from Dr. Hugo Pequeno Monteiro (UNIFESP).C. sylvestris, as well as pure α-zingiberene, zingiberene partially hydrogenated and zingiberane, were resuspended in dimethyl sulfoxide (DMSO) at a final concentration of 10 mg/mL, diluted in RPMI medium containing 10% fetal calf serum ranging from 100 to 0 µg/mL and incubated with 1 × 104 cells in a 96-well plate. After 18 h of incubation, cell viability was measured using the Cell Proliferation Kit I (MTT) , an MTT-based colorimetric assay [The essential oils extracted from leaves of ic assay . ReadingC. sylvestris analyzed in this work showed a different profile in comparison to other previously analyzed oils, being composed by approximately 50% of α-zingiberene. The cytotoxic evaluation displayed that the crude essential oil was active against all cell lines tested, while purified α-zingiberene showed less activity, indicating that there may be synergism of zingiberene with other substances present in the crude oil for activity. For PHZ, the cytotoxic activity decreased, while THZ showed cytotoxic activity against almost all the tested cell lines. These findings suggests that oil from C. sylvestris may be an interesting agent for cancer treatment; however additional studies must be done including isolation of active compound, mechanism of action on tumor cells as well as experimental assays.Essential oil from leaves of


Neurofibromatosis 1 and 2, although involving two different tumour suppressor genes , are both cancer predisposition syndromes that disproportionately affect cells of neural crest origin. New therapeutic approaches for both NF1 and NF2 are badly needed. In promising previous work we demonstrated that two non-steroidal analogues of 2-methoxy-oestradiol (2ME2), STX3451-7-methoxy-6-sulfamoyloxy-1,2,3,4-tetrahydroisoquinoline), and STX2895 -1,2,3,4-tetrahydroisoquinoline) reduced tumour cell growth and induced apoptosis in malignant and benign human Neurofibromatosis 1 (NF1) tumour cells. In earlier NF1 mechanism of action studies we found that in addition to their effects on non-classical hormone-sensitive pathways, STX agents acted on the actin- and myosin-cytoskeleton, as well as PI3Kinase and MTOR signaling pathways. Tumour growth in NF2 cells is affected by different inhibitors from those affecting NF1 growth pathways: specifically, NF2 cells are affected by merlin-downstream pathway inhibitors. Because Merlin, the affected tumour suppressor gene in NF2, is also known to be involved in stabilizing membrane-cytoskeletal complexes, as well as in cell proliferation, and apoptosis, we looked for potentially common mechanisms of action in the agents’ effects on NF1 and NF2. We set out to determine whether STX agents could therefore also provide a prospective avenue for treatment of NF2.STX3451 and STX2895 were tested in dose-dependent studies for their effects on growth parameters of malignant and benign NF2 human tumour cell lines in vitro. The mechanisms of action of STX3451 and STX2895 were also analysed.Although neither of the agents tested affected cell growth or apoptosis in the NF2 tumour cell lines tested through the same mechanisms by which they affect these parameters in NF1 tumour cell lines, both agents disrupted actin- and myosin-based cytoskeletal structures in NF2 cell lines, with subsequent effects on growth and cell death.Both STX3451 and STX2895 provide new approaches for inducing cell death and lowering tumour burden in NF2 as well as in NF1, which both have limited treatment options. Both Neurofibromatosis 1 and 2 (NF1 and NF2) are disorders characterized by the formation of tumours of the peripheral and central nervous system , 4.DCAF1 [Neurofibromatosis type II (NF2) is associated with loss-of-function mutations in the NF2 gene that encodes the multi-functional protein, Merlin (Moesin-Ezrin-Radixin-like protein) , also knDCAF1 .NF2 affects one in 25,000–30,000 live births worldwide. A hallmark of the disease is the formation of bilateral vestibular Schwannomas, as well as the formation of multiple meningiomas, extramedullary spinal tumours, and ependymomas . UncontrCurrent treatment options for NF2 tumours include surgical resection of either part of or the complete tumour, which is difficult to perform without damaging nerves. Stereotactic radiosurgery is also an option, however the risk of malignant transformation rises several years post-surgery , 21. AltPrevious studies from our laboratories  demonstr50 of 40 and 52 nM on DU-145 human prostate and MDA MB-231 breast cancer cell lines, respectively [The two non-steroidal 2ME2 analogues, STX3451 -7-methoxy-6-sulfamoyloxy-1,2,3,4-tetrahydroisoquinoline) and STX2895 -1,2,3,4-tetrahydroisoquinoline) . Recentectively .Fig. 1CHere, we show that NF2 status plays a role in the effectiveness of these non-steroidal 2ME2 derivatives on cell viability in vitro. STX2895 and STX3451 treatment on NF2 null (−/−) cell lines induces nuclear fragmentation, microtubular disruption, inhibition of cellular migration/wound healing, and can induce apoptosis in both benign and malignant NF2 tumour-derived cells.−/− cell line from a 41-year-old woman [+/+ cell line derived from the brain of 61-year-old man [2 incubator [NF2-null malignant meningioma KT21-MG1-Luc5D cells  and theld woman ) were geld woman ) were ki-old man ) was obtSTX3451 and STX2895 were synthesized according to previously published protocols , 45. Pak4/well) determined by a hemocytometer. STX3451 or STX2895 at concentrations of 100 nM, 300 nM, 600 nM, and 1000 nM was dissolved in vehicle  and were added to the wells 2 hours after plating the cells. The final concentration of DMSO was 1%. PF3758309 (PF) and Frax1036 concentrations ranged from 0.03 μM to 20 μM and from 2.5 μM to 25 μM, respectively. The final concentration of DMSO was 2.5%. STX3451, STX2895, PF, or Frax1036 was replenished after 2 days of culture. Cell growth was assessed using CellTiter 96  [Cells were plated in 96-well Primaria™ plates  , 48.40,000–80,000 cells plated on 0.1% porcine gel-coated coverslips were treated with DMSO, STX3451, STX2895, PF3758309, or Frax1036 for various time periods. Immunocytochemistry and counter-staining with DAPI , were carried out as reported previously [5 cells were plated in a 10 mm cell culture dish and allowed to grow to confluence. Wounds were made using a ruler to maintain a straight edge and a 200 μl pipette tip applied to the bottom of the dish to remove the cells. The culture plates were then washed twice with PBS. Fresh medium with DMSO, STX3451, or STX2895 was added to the dishes. STX3451 and STX2895 were used at 300 nM for KT21, Ben-Men-1, and HEI-193 cells, 1000 nM for IOMM-Lee and BJ cells. Photographs of the wounds and subsequent cell migration and “wound filling” were taken using a Nikon SMZ1500 dissecting microscope and digital camera  at the time points indicated. The dimensions of the “wound” were measured with ImageJ software.The ability of cells to migrate was assessed by wound-healing assays , 50. 2 ×Cell lysates were collected after 24 h treatments with DMSO, STX3451, STX2895, PF, or Frax1036. Western blotting was performed as described in the studies on NF1 . MembranThe STX therapeutic agents tested are multifaceted in their mechanisms of action. This study was directed at discovering whether the mechanisms of action that make the STX agents promising potential therapeutics for the treatment of NF1 would also apply to the potential treatment of NF2. Our previous studies concluded that the agents’ effects on the growth of and induction of apoptosis in human NF1 tumour cells were both through NF1-specific pathways and through significant effects on both the actin-based and myosin-based cytoskeleton . The sam−/− benign meningioma cells), CH157MN (NF2−/− meningioma cells), HEI-193 (NF2−/− benign vestibular Schwannoma cells), KT21  and IOMM-Lee  to the individual agents or to a combination of the two. Our results showed that both agents either completely blocked cell proliferation or induced cell death in NF2−/− cells whether the cell lines were derived from benign or malignant tumours  testing whether the STX agents act mechanistically on the same pathways on which we found them to be very effective on growth and apoptosis in human NF1 tumour cell lines, we currently have been able to detect NO significant effects of either of the agents on the growth, morphology of NF2 cell lines through the pathways that are known to be important for the growth of or cell death in NF1 human tumour cell lines . This iOur previous studies on NF1 cell lines demonstrated that STX3451 caused nuclear fragmentation in both the NF1-null malignant tumour cell line ST88 and in benign plexiform neurofibroma (PNF) cells, although this result was less significant for PNF cells .We therefore examined whether this effect was also observed in our studies of NF2-null tumour cells. DAPI staining revealed that treatment with both STX3451 and STX2895 resulted in a dramatic increase in nuclear fragmentation, which was seen in both KT21 and Ben-Men-1 cell lines 48 h after treatment; with STX3451being more effective than STX2895  were apoptotic at 72 h, indicating that these analogues of 2ME2 also have effects on cell growth/apoptosis in the benign Ben-Men-1 cells. However, the effects seen are much less than those seen when either STX agent was used to treat malignant KT21 cells. By contrast, 72 h treatment with PF resulted in 20% annexin V-positive cells at very low concentration, the IC50 for KT21 was 29.25 nM . Frax103Finally, STX3451 and STX2895 at 1000 nM did not significantly increase the percentage of annexin V-positive BJ cells at 72 h, consistent with the results seen in the cell viability assays (discussed above). At concentrations that effectively induced apoptosis in NF2 tumour cells, STX3451 and STX2895 did not cause such programmed cell death in BJ fibroblasts. PF at 5 μM also did not increase the percentage of annexin V-positive BJ cells, whilst Frax1036 at 10 μM induced 55% cells to go through apoptosis.STX3451 has been demonstrated to depolymerize microtubules in both NF1 ST88 and PNF cell lines . Our curOur results from annexin V and α-tubulin staining Fig.  suggest . We found that the cell lines, which we evaluated migrated to fill the wound gaps at different rates, and that STX3451 and STX2895 inhibited wound healing in the NF2 cells with different kinetics. The untreated control Ben-Men-1 cells were able to fill in the gaps by 4 days after the wound was made. However, cells treated with either STX3451 or STX2895 also filled in the gap, but at a much slower pace  [We previously demonstrated that STX3451 induces apoptosis in human NF1-deficient ST88 and S462 malignant peripheral nerve sheath tumour (MPNST) cell lines at very low concentration 300 nM), whilst arresting cell proliferation in an NF10 nM, whiIntriguingly, our results showed that STX3451 and STX2895 were not as effective against a permanent NF2 wild type malignant meningioma cell line, IOMM-Lee, indicating that NF2 status could be important for the effects of these compounds. Our results also showed that these 2ME2 analogs were more potent than Pak inhibitors in inducing cell death. However, their mechanistic effects on the cells were different: STX3451 and STX2895 affect microtubules  and/or meningiomas has not been well-studied. It has been shown that in sporadic VS, both oestrogen receptor (ER) and progesterone receptor (PR) were up-regulated, whilst in NF2-related VS, PR was down-regulated [Our previous study showed that the non-steroidal analog of 2ME2, STX3451, caused microtubule depolymerization and cell death in NF1 deficient tumour cells, even in the presence of elevated hormones . Whetheregulated . Amongstegulated . PR-negaegulated , and expegulated . Howeveregulated , 61 and egulated . Treatmeegulated . TherefoSince Merlin affects several key signalling pathways in the cytoplasm and nucleus, including PI3K signaling, mTORC1, the RAS and Src pathways, the Hippo kinase cascade, and CRL4-DCAF , inhibitSo far, none of the drugs tested in vitro or in pre-clinical or clinical trials has been effective on NF2 meningioma. Although, another mTOR inhibitor, AZD-2014, and a combination of smoothened receptor inhibitor, vismodegib, and FAK inhibitor, vistusertib, are in phase II trials. AR-42, a histone deacetylase inhibitor targeting CRL4-DCAF, which induced meningioma cell apoptosis in vitro and in xenografts, is also in phase I trials. Even if some inhibitors for NF2/Merlin signalling pathways have promising efficacy, drug resistance to these inhibitors may eventually occur and alternative choices are necessary. The microtubule-disrupting non-steroidal 2ME2 analogs could provide another option for treating NF2-related tumours, both vestibular Schwannomas and meningiomas, either alone, or in conjunction with other agents. Pre-clinical studies using several similar THIQ-based molecules have been shown to be safe and efficacious for reducing the size of human breast cancer  and melaBoth STX3451 and STX2895 provide new approaches for inducing cell death and lowering tumour burden in NF2 as well as in NF1, which both have limited treatment options.


Skeletonema marinoi have selective cytotoxic activity against the haematological cancer cell line U-937 and colon cancer cell line HCT-116 compared to normal MePR-2B cells. LC-MS analysis of the raw extract revealed that in their natural form, MAGs occur as 2-monoacyl derivatives and include mainly C16 and C20 analogues, but they are converted into the corresponding 1-isomers during purification processes. Pure compounds along with the synthetic 1-monoarachidonoylglycerol tested on HCT-116 and U-937 tumor cell lines induced cell death via apoptosis. The mechanism of action was investigated, and we show that it involves the induction of apoptosis through caspase 3/7 activation. These findings pave the way for the possible use of these molecules as potential anticancer agents or as precursors for the generation of new and more potent and selective compounds against tumor cells. Microalgae are an excellent source of valuable compounds for nutraceutical and cosmeceutical applications. These photosynthesizing microorganisms are amenable for large-scale production, thus overcoming the bottleneck of biomass supply for chemical and activity characterization of bioactive compounds. This characteristic has recently also prompted the screening of microalgae for potential pharmaceutical applications. Here, we show that monoacylglycerides (MAGs) purified from the marine diatom  Chlorella ovalis and the dinoflagellate Amphidinium carterae suppressed the growth of HL-60 cells (human promyelocytic leukemia cell line) +.1-mono-


The use of cell lines in research can be affected by cell line misidentification. Short tandem repeat (STR) analysis is an effective method, and the gold standard, for the identification of the genetic origin of a cell line, but methods that allow the discrimination between cell lines of the same genetic origin are lacking. Here, we use intact cell MALDI-ToF mass spectrometry analysis, routinely used for the identification of bacteria in clinical diagnostic procedures, for the authentication of a set of cell lines consisting of three parental neuroblastoma cell lines  and eleven drug-adapted sublines. Principal component analysis (PCA) of intact-cell MALDI-ToF mass spectrometry data revealed clear differences between most, but not all, of the investigated cell lines. Mass spectrometry whole-cell fingerprints enabled the separation of IMR-32 and its clonal subline IMR-5. Sublines that had been adapted to closely related drugs, for example, the cisplatin- and oxaliplatin-resistant UKF-NB-3 sublines and the vincristine- and vinblastine-adapted IMR-5 sublines, also displayed clearly distinctive patterns. In conclusion, intact whole-cell MALDI-ToF mass spectrometry has the potential to be further developed into an authentication method for mammalian cells of a common genetic origin. Cell line misidentification has been a major issue since the first mammalian cell lines were established ,2. HeLa,While there are established methods for the determination of the genetic origin of cell lines, methods for the discrimination of cell lines derived from the same genetic origin (isogenic cell lines), such as clonal sublines, different cell lines derived from the same organism, or sub-cell lines that were adapted to growth under specific conditions are currently under development. Intact-cell MALDI-ToF mass spectrometry is routinely used for the identification of bacteria in clinical diagnostic procedures  and has One group of cell lines where STR methods are not able to distinguish between them is drug-adapted cancer cell lines, which are used as in vitro models of acquired drug resistance. The ATP-binding cassette (ABC) transporters ABCB1  and ABCC1 , major mediators of drug resistance in cancer, were discovered in drug-adapted cell lines ,32. More5 cells [17 in Caucasian- and Hispanic-American populations. The genetic origin of all cell lines was successfully confirmed  could discriminate between different cell lines. First, we looked at the parental cell lines IMR-32, IMR-5 and UKF-NB-3 using the comparisons PC1 vs. PC2, PC1 vs. PC3 and PC2 vs. PC3. The comparison PC2 vs. PC3 showed the largest separation . NotablyrCDDP1000) and oxaliplatin (UKF-NB-3rOXALI2000). It further separated the two sublines that had been adapted to closely related platinum drugs , topotecan (IMR-32rTOPO8) and melphalan (IMR-32rMEL500) displayed clearly distinctive patterns. Only the oxaliplatin-resistant IMR-32 subline (IMR-32rOXALI800) was difficult to differentiate from the parental IMR-32 cells  and the cisplatin-adapted (IMR-5rCDDP1000) subline formed clearly distinctive clusters (rDOCE20), dacarbazine-resistant (IMR-5rDACARB40) and vincristine-resistant (IMR-5rVCR10) sublines were harder to distinguish, although they also formed individual clusters  did not cluster closely together with regard to their mass spectrometry profiles. Further research will have to show whether this observation has functional implications.When directly comparing the STR and intact-cell MALDI-ToF analysis, as stated above, the STR approach showed four of the eleven drug-adapted sublines displayed identical STR profiles to the respective parental cell lines and the other sublines showed one to four changes, and hence all sublines displayed a >80% match with the respective parental cell lines, regarded as the threshold for authenticity  and hencIn conclusion, intact-cell MALDI-ToF mass spectrometry is a promising technique for the authentication of mammalian cells of a common genetic origin, but further development is required before it can be used as a reliable, generally applicable method, either on its own or (which seems to be more likely for the foreseeable future) in combination with other established methods, in particular STR typing. This approach represents a rapid method of authenticating cell lines that could complement traditional STR profiling. In developing this approach to authentication, we recommend that a minimum of three replicate analyses be undertaken for each sample to account for technical variation, and ideally three biological samples would also be analysed. We also suggest that a bank of quality controls consisting of historical fingerprints of authenticated cell lines be curated alongside frozen samples of authenticated cells that could be analysed alongside naive samples to account for any day-to-day variation in analyses and to give further confidence to identification of unknown cell lines. Further research will have to (1) establish that the methodology is able to identify unknown cell lines using statistically robust validation approaches and by masking the identification of cell lines to experimentalists, (2) show the extent to which cross-contaminations can be detected based on mass spectrometry profiles, (3) investigate the potential influence of cell culture conditions on mass spectrometry profiles and (4) establish databases with cell line profiles and quality standards for wider application by the community. Notably, methods for the authentication of cell lines of the same origin will also be important for areas beyond cancer research. For example, mouse strains are often derived from inbred strains and can also not be distinguished by STR ,59.The MYCN-amplified neuroblastoma cell line UKF-NB-3 was established from a bone marrow metastasis derived from a stage 4 neuroblastoma patient . The MYCrCDDP1000) or oxaliplatin (UKF-NB-3rOXALI2000), IMR-32 sublines adapted to carboplatin (IMR-32rCARBO1000), melphalan (IMR-32rMEL500), oxaliplatin (IMR-32rOXALI800) or topotecan (IMR-32rTOPO8), and IMR-5 sublines adapted to cisplatin (IMR-5rCDDP1000), dacarbazine (IMR-5rDACARB40), docetaxel (IMR-5rDOCE20), vinblastine (IMR-5rVINB20) or vincristine (IMR-5rVCR10) were established by continuous exposure to stepwise increasing drug concentrations, as previously described [https://research.kent.ac.uk/ibc/the-resistant-cancer-cell-line-rccl-collection/).UKF-NB-3 sublines with acquired resistance to cisplatin  supplemented with 10% foetal calf serum (FCS), 100 IU/mL penicillin and 100 µg/mL streptomycin at 37 °C. Cells were routinely tested for mycoplasma contamination.Cell lines were authenticated with the current STR typing technique of the DSMZ , which, according to the STR Typing Standard (ASN-0002: Human cell line authentication: STR profile standardization), uses an expanded set of STR markers that goes beyond the standard set of eight markers .Amplification was performed in DNase- and RNase-free vials of 0.2 mL in an i-Cycler . Per sample, the master mix contained 10 pmol total primer solution (1 µL of a 10 µM solution), 2.5 µL 10 x hot start PCR buffer (any supplier), 1 µL dNTP solution (5 µM), 0.2 µL hot start Taq polymerase solution , 19.5 µL distilled water and 1 µL genomic DNA solution (10 ng/µL). The genomic DNA was added last and independent from the master mix. The PCR programme was carried out as follows: 95 °C for 3 min, 1 repeat; 94 °C for 30 s, 57 °C for 30 s, 72 °C for 45 s, 30 repeats; 60 °C for 15 min, 1 repeat.For fragment detection and generation of allelic STR lists, aliquots of 1 µL of PCR products were combined with 0.25 µL of an internal size standard  in a total volume of 30 µL of sample loading solution on a microtiter plate. The samples were loaded automatically and analysed by the capillary electrophoresis system CEQ 8000 (Beckman-Coulter) using established fragment analysis parameters. Generated STR profiles were submitted to search within a reference STR database of cell lines . Additio4 viable cells were transferred to a 96-well plate format rack, centrifuged for 5 min at 960 rcf, and the supernatant removed. After washing by resuspension and centrifugation in 0.5 mL of PBS and in 0.35 M sucrose, cell pellets were stored at –80 °C until analysed.The intact-cell MALDI-ToF mass spectrometry analysis was performed essentially as previously described . 6.25 × 20 mg/mL (saturated) solution of sinapinic acid was dissolved in matrix buffer ) and sonicated in a water bath for 15 min before centrifugation. After removal from –80 °C storage, cells were allowed to equilibrate to room temperature for 15 min prior to resuspension in 50 µl of sinapinic acid solution and incubation at 4 °C for 3 h. Then, 1 µl of each sample was spotted onto a 384 MTP ground steel MALDI-ToF plate  and air-dried, before the plate was placed into the MALDI-ToF mass spectrometry instrument (Bruker Ultraflex).n = 3) biological cultures were analysed, each culture analysed by triplicate technical analyses of the samples.Spectra were collected using the following MALDI-ToF ms instrument settings: laser frequency: 20 Hz; polarity +ve; ion sources: 1. 20 kV, 2. 17.25 kV, lens 5.0 kV; gating mode: maximum strength; suppress @ 4000 Da; pulsed ion extraction 550 nS; range 5000–60,000 Da; sample rate 0.1 Gs/s; resolution enhanced 100 mV electronic gain; smooth high. For each sample, 100 shots were summed and saved. In all cases, triplicate (m/z vector); 2. Baseline Correction (removes the effect of noise introduced by the matrix); 3. Filtering ; 4. Alignment ; 5. Quality Control ; 6. Normalisation . Details for all steps have been outlined previously in [http://tinyurl.com/msresample). In addition, a baseline correction was performed using the “msbackadj” function in the MATLAB Bioinformatics Toolbox (http://tinyurl.com/msbackadj). Peak alignment was performed using the “msalign” function from the MATLAB Bioinformatics Toolbox . Finally, data was normalised using the “msnorm” function from the MATLAB Bioinformatics Toolbox , as described in [Data files were exported from the Bruker Flex Analysis software in ASCII format for preprocessing in MATLAB (Version R2017a for Windows) prior to the application of the principal component analysis as described in . The folously in . Resamplribed in .


Dasatinib (Sprycel) was developed as a tyrosine kinase inhibitor targeting Bcr-Abl and the family of Src kinases. Dasatinib is commonly used for the treatment of acute lymphoblastic and chronic myelogenous leukemia. Previous clinical studies in melanoma returned inconclusive results and suggested that patients respond highly heterogeneously to dasatinib as single agent or in combination with standard-of-care chemotherapeutic dacarbazine. Reliable biomarkers to predict dasatinib responsiveness in melanoma have not yet been developed.Here, we collected comprehensive in vitro data from experimentally well-controlled conditions to study the effect of dasatinib, alone and in combination with dacarbazine, on cell proliferation and cell survival. Sixteen treatment conditions, covering therapeutically relevant concentrations ranges of both drugs, were tested in 12 melanoma cell lines with diverse mutational backgrounds. Melanoma cell lines responded heterogeneously and, importantly, dasatinib and dacarbazine did not synergize in suppressing proliferation or inducing cell death. Since dasatinib is a promiscuous kinase inhibitor, possibly affecting multiple disease-relevant pathways, we also determined if basal phospho-protein amounts and treatment-induced changes in phospho-protein levels are indicative of dasatinib responsiveness. We found that treatment-induced de-phosphorylation of p53 correlates with dasatinib responsiveness in malignant melanoma.Loss of p53 phosphorylation might be an interesting candidate for a kinetic marker of dasatinib responsiveness in melanoma, pending more comprehensive validation in future studies. Dasatinib (N-(2-chloro-6-methyl-phenyl)-2-((6-(4-(2-hydroxyethyl)-piperazin-1-yl)-2-methylpyrimidin-4-yl)amino)-1,3-thiazole-5-carboxamide) is an orally active tyrosine kinase inhibitor developed to target Bcr-Abl and the family of Src kinases . LargelyDasatinib has been and still is being tested as a single agent and in combination therapies, for example together with other kinase inhibitors or genotoxic chemotherapeutics, in haematological cancers and solid tumours. At the time of writing, clinicaltrials.gov lists 72 active and planned studies. In melanoma, the therapeutic efficacy of dasatinib was tested as a single treatment as well as in combination with the standard-of-care chemotherapeutic dacarbazine (DTIC) in phase I/II trials. In a phase I trial with 29 patients, the reported objective response rate (ORR) was 13.8%, and the 6-month progression-free survival (PFS) was 20.7%. In a phase II trial with 36 patients, the ORR was 5%, and the 6-month PFS was 13%. . With thDasatinib and dacarbazine both were reported to impair cell proliferation, either by kinase inhibition or by DNA damage-induced cell cycle arrest , 12. To 72 h after treatment commenced, heterogeneous responses to dasatinib or dacarbazine on cell proliferation were observed. For dasatinib single agent treatment, WM115 was the most sensitive cell line, with 10–100 nM sufficient to reduce proliferation to approximately 25% ), a target associated with c-Src signaling, directly affected by dasatinib and also known for being implicated in downstream RAS/RAF/MEK/ERK signaling, thereby affecting cell growth and proliferation [97), a taBasal phospho-protein expression across the melanoma cell line panel was highly heterogeneous Fig. a. ComparHere, we studied the responsiveness of melanoma cells to dasatinib, alone or in combination with standard-of-care chemotherapeutic dacarbazine, by systematically analyzing growth inhibition and cell death at therapeutically meaningful concentrations ranges of both drugs. To our knowledge, this is the most comprehensive quantitative analysis of the effect of these therapeutics on melanoma cell proliferation and viability to date. Melanoma cells responded heterogeneously, with both dasatinib-responsive and highly dasatinib-resistant cell lines identified. Of thirteen phosphoproteins studied, all of which are directly or indirectly involved in regulating cell growth, proliferation, or cell cycle progression, none correlated with dasatinib responsiveness in their expression amounts. Interestingly though, the amounts of phosphorylated p53 (S46) dropped significantly in dasatinib-responsive cell lines following treatment, highlighting this response as a previously unknown kinetic marker candidate for susceptibility to dasatinib. Determining the specificity of such a marker more accurately, however, would require further studies, since p53 phosphorylation also dropped in one non-responsive cell line (WM3060).p = 0.06), indicating that modulators of Src activity might provide scope for marker development [Dasatinib has been studied comprehensively for its spectrum of kinase targets. Dasatinib inhibits diverse kinases besides the Src family at therapeutically meaningful concentration ranges. These targets include FAK, BTK, EPHs and p38 MAPK and others . It therelopment . c-KIT, elopment , can be elopment , 10, 32.Instead of studying pre-treatment markers, an alternative approach lies within investigating if kinetic markers for dasatinib responsiveness can be identified. We found that the amounts of p53 (S46) drop upon dasatinib treatment in responsive melanoma cell lines. Among other kinases, the direct dasatinib target p38 MAPK can phosphorylate p53 at S46 . p53 is w/v) heat inactivated fetal bovine serum (Sigma-Aldrich), 100 U/ml penicillin and 100 mg/ml streptomycin (Sigma-Aldrich). Cells were grown at 5% CO2 and 37 °C. To study treatment responses, cells were seeded into flat bottom 96 well plates and treated with dasatinib , dacarbazine  or combinations of both drugs. Drugs were added simultaneously.Cells were obtained as authenticated stocks from the ATCC  , the Wistar Institute   and the DSMZ  (Mel Juso). Five cell lines carried activating BRAF mutations , four cell lines NRAS mutations , one cell line a c-KIT mutation (WM3211), and two cell lines were BRAF/NRAS/c-KIT wildtype . Cells were cultured in DMEM  supplemented with 4 mM L-glutamine, 4.5 g/l glucose, 10% . Measurements were performed on a BD LSRII SORP HTS cytometer . Data files were exported in .fcs format and analyzed using Flowing Software . All experiments were independently repeated at least three times and all treatment conditions were measured in triplicates.Cells were seeded into 96 well plates and lysed after 24 h treatment with dasatinib. The protein concentrations in cell lysates were adjusted to 300 μg/ml, using the BCA assay kit for protein concentration determination (Pierce). xMAP bead-based ELISA type assays  were performed on a Luminex FLEXMAP 3D® instrument using a custom developed phosphoprotein 13-plex panel : RAC-alpha serine/threonine-protein kinase (AKT) - S473, Focal adhesion kinase 1 (FAK1) - Y397, Transcription factor AP-1 (c-JUN) - S63, dual specificity mitogen-activated protein kinase kinase-1 (MEK1/MP2K1) - S217/S221, Mitogen-activated protein kinase-3 (ERK1/MK03) - T202/Y204, Mitogen-activated protein kinase 9 (JNK2/MK09) - T202/Y204, Tyrosine-protein phosphatase non-receptor type 11 (PTN11/SHP2) - Y542, Cyclic AMP-responsive element-binding protein 1 (CREB) - S133, NF-κB inhibitor alpha (IκBa) - S32/S36, Ribosomal protein S6 kinase beta-1 (KS6B1/P70S6K) - T389, Cellular tumor antigen p53 (P53) - S46, Signal transducer and activator of transcription 3 (STAT3) - Y705, Serine/threonine-protein kinase WNK1 (WNK) - T60. For normalization, the levels of total GAPDH protein were analyzed.Melanoma cell lines were homogenized in lysis buffer  with addition of PhosSTOP phosphatase inhibitor cocktail and cOmplete protease inhibitor cocktail . Total protein concentrations of samples were determined by Bradford protein assay . Samples were loaded (30 μg) and separated on Bolt 4–12% Bis-Tris Plus Gels  at 150 V, 400 mA for 40 min. Proteins were transferred to nitrocellulose membranes using the iBlot 2 gel transfer device . The membranes were blocked in 5% BSA in TBST for 1 h at room temperature and incubated with primary antibodies overnight at 4 °C. The following primary antibodies were used: rabbit-anti p53 , anti-mouse beta-Actin  and rabbit-anti phospho-p53 (S15) , anti-rabbit p38 MAPK , anti-mouse phospho-p38 MAPK (T180 / Y182) , anti-mouse alpha-Tubulin ,. The membranes were washed 3 × 10 min in TBST and incubated with secondary antibody HRP goat-anti rabbit  for 1 h on a room temperature followed by 3 × 10 min wash in TBST. Luminata Forte Western HRP substrate  was added and chemiluminescence signal was detected on Amersham Imager 600 system . Signal intensities above background were quantified using ImageJ software.GraphPad Prism  was used for statistical analysis.


Both NGR and RGD conjugated forms of ketoprofen showed higher cytotoxic activity against OVCAR3 and HT-1-80 respectively.It is well known that Arginine-Glycine-Aspartic acid (RGD) and Asparagine-Glycine-Arginine (NGR) peptides preferentially bind to integrin receptors and aminopeptidase N respectively and these two receptors play important roles in angiogenesis. Therefore ketoprofen as a non-selective cox Inhibitor was conjugated with linear RGD and NGR to take advantage of targeting capability of these two motifs and delivering ketoprofen to these cancer cells with overexpression of integrin and aminopeptidase N. In order to investigate the impact of possible steric hindrance due to the attachment of the drug to the peptide, a linear six carbon (hexanoic acid) linker was also used as a spacer. Cytotoxic effect of the synthesized compounds was evaluated against a group of cancer cell lines, including MCF-7, A2780 (α Cancer is unusual cell growth and division which can spread to different parts of body. Chemotherapy is a main approach for cancer treatment and limited by low selectivity of chemotherapy drugs for normal tissues, especially tissues with rapidly cells division and growth.v integrin is highly overexpressed on activated endothelial of cancer cells and tumor cells, but it is not expressed in resting endothelial cells and most normal organ systems, making it a potential target for antiangiogenic strategy in cancer therapy  and NGR (Asparagine-Glycine-Arginine) motifs have been recognized as targeting tools based on phage display studies . RGD is er cells . Since αer cells -19, RGD er cells . NGR pepet al. have demonstrated that ketoprofen inhibits human colon tumor cell N-acetyltransferase (NAT) activity, gene expression and DNA adduct formation for the first time in 2006  which inhibits COX-1 and COX-2 enzymes. Numerous studies demonstrate that NSAIDs have anticancer properties and reduced the risk of cancers like colon and liver  Several  in 2006 . Marjano in 2006 . Based oMaterials and methodsa-9-Fluorenylmethoxycarbonyl (Fmoc)-protected amino acids, Rink amid resin, 1-[Bis(dimethylamino)methylene]-1H- 1,2,3-triazolo pyridinium 3-oxid hexafluorophosphate (HATU) were purchased from GL Biochem Company (Shanghi) Ltd. 1HNMR spectra were recorded on a 500 MHz Bruker spectrometer. ESI-MS spectra were obtained by Agilent 6410 Triple Quad LC/MS. IR spectra were recorded on a PerkinElmer IR spectrophotometer as potassium bromide discs. The purity of compounds was confirmed by thin layer chromatography using Whatman Sil All chemicals and solvents were purchased from Merck . N® HR ODS column .G/UV254 silica gel plates as the stationary phase and with suitable mobile phase with fluorescent indicator, and the spots were visualized under 254 and 366 nm illumination. All products was purified by reverse phase HPLC on a Knaur chromatography system equipped with a Waters Prep Nova-PakGeneral procedure for synthesis RGD conjugated with ketoprofen2 gas for 20 min. The completion of each coupling reaction and fmoc deprotection was tested by Kaiser Test which is the reaction of ninhydrin with amines. Rink amide resin (0.5 g) was placed in a reactor and suspended in DMF under nitrogen atmosphere for 15 min. The fmoc group was removed with 20% piperidine in DMF (3 × 5 mL). After washing with DMF, coupling reaction was conducted. For each amino acid coupling: 2eq AA, 2eq HATU, 4eq N,N-diisopropylethylamine (DIEA), 15 mL DMF was mixed and added to the resin. The mixture was purged with NAfter the last amino acid coupling reaction and removal of fmoc protecting group, the ketoprofen was coupled to the free amino group of the peptide, using the same standard procedure which is used for the coupling of amino acids. Finally resin washed and dried then linear peptide cleaved with trifluoroacetic acid (TFA) cocktail: TFA: Thioanisol: p-cresol: triisopropylsilane (TIS) (84%: 12%: 2%: 2%), during 2 h at room temperature. The reaction mixture was filtered and cold Diethyl Ether (50 mL) was added to the filtrate. The precipitate thus obtained was separated using centrifugation.RGD 3CN in H2O+ 0.1% TFA); 1H NMR  δ: 1.54 , 1.70 , 2.63-2.69 , 3.11 , 3.86 , 4.50-4.53 , 7.15-8.68 ppm ; IR (KBr): υ (cm-1) = 1656, 3330; LC-MS (ESI) m/z (M + H)+ calcd for C12H23N7O5: 346.0, found: 346.1. The title compound (50 mg) was obtained as a white solid with 63% yield at 97% purity ; 1H NMR  δ: 1.50 , 1.99 , 2.5 , 2.58-2.69 , 3.23 , 4.03 , 7.15-8.3 ppm ; IR (KBr): υ (cm-1) = 1656, 3330; LC-MS (ESI) m/z (M + H)+ calcd for C12H23N7O5: 346.0, found: 346.1.The title compound (57 mg) was obtained as a white solid with 64% yield at 93% purity ; 1H NMR  δ: 1.32-1.33, 1.45-1.46 , 1.64-1.82 , 2.5 , 2.96,3.07 , 3.64-3.69 , 3.82-3.85 , 4.15-4.16 , 4.26-4.27 , 4.38-4.43 , 7.05-8.31, 11.8  ppm; IR (KBr): υ (cm-1) 1656, 3330 ; LC-MS (ESI) m/z (M + H)+ calcd for C28H35N7O7: 581, found: 582.2. The title compound (83 mg) was obtained as a white solid with yield 65%, 98% purity 4-CO-RGD3CN in H2O+ 0.1% TFA); 1H NMR  δ: 1.05 , 1.20-1.23 , 1.28 , 1.48 , 1.7 , 2.07 , 2.8 , 3.17 , 3.38 , 3.46-3.52 , 4.16 , 4.55 , 5.88-5.93 , 7.00-7.8 ppm , IR (KBr): υ (cm-1) 1654, 3356 ; LC-MS (ESI) m/z (M + H)+ calcd for C34H47N9O7: 693, found: 694.2.The title compound (103 mg) was obtained as a white solid with yield 65%, 98% purity ; 1H NMR  δ: 1.05 , 1.25 , 1.45 , 2.5 , 2. 6 , 3.47 , 3.6 , 3.9 , 4.16 , 4.43 , 7.05-8.29 , 11.8  ppm; IR (KBr): υ (cm-1) 1656, 3330; LC-MS (ESI) m/z (M + H)+ calcd for C28H36N8O7: 581, found: 582.2. The title compound (73 mg) was obtained as a white solid with yield 57%, 98% purity 4-CO-NGR3CN in H2O+ 0.1% TFA); 1H NMR  δ: 1.05 , 1.19-1.25 , 1.46-1.48 , 1.64 , 2.28 , 2.49 , 2.90 , 2.97 , 3.40 , 3.46-3.51, 4.12 , 4.8  5.88-5.93 , 7.00-8.03 ppm ; IR (KBr): υ (cm-1); IR (KBr): υ (cm-1) 1659, 1647, 3331; LC-MS (ESI) m/z (M + H)+ calcd for C34H47N9O7: 693, found 694.1. The title compound (110 mg) was obtained as a white solid with yield 69%, 97% purity , OVCAR-3 (high αVβ3), SKOV-3 (low CD13), HT-1080 (high CD13) and MCF-7 and fibroblast cell were selected. A2780 and MCF-7 are both cancer cells, but A2780 is αv β3 integrin overexpressing cell line. Three concentrations of products were prepared and were evaluated after 48 h by using MTT method. Therefore cells cultured in culture media (RPMI1640) at 37 °C under 5% CO2 supplemented with 10% fetal bovine serum (FBS), 100 U/mL penicillin and 100 μg/mL streptomycin. Nearly 104 cells were seeded into 96-well plates and were grown for 24 h in incubator. Compounds and doxorubicin added to wells, cells were incubated at 37 °C for 48 h. Cells without any adding of compound were used as negative control, Doxorubicin was used as positive control and DMSO as the solvent of the test compounds. After 48 h, 10 μL of MTT (5 mg/mL in PBS) was added at 37 °C for 4 h. The medium with MTT was removed and 100 μL DMSO was added to each well, cover the plate from light, shake it for 5 min, then optical densities were read at 570 nm with a reference wavelength of 630 nm as background using a spectrophotometer plate reader .In order to evaluate the cytotoxic activity of the synthesis compounds, five cell lines: A2780 (low αMolecular modeling (docking) studies vβ3 integrin receptor in the presence of the Mn2+ ion and with active sites of APN. The crystal structure of the extracellular domain of the αvβ3 integrin receptor in the presence of the Mn2+ ion (PDB entry code: 1L5G)    was obtae: 2DQM) .vβ3 integrin receptor. The quality of the docked structures was assessed by measuring the intermolecular energy of the ligand-enzyme assembly. Grid map dimensions for 2DQM (71.9186 × 47.8031 × 7.3077) were set surrounding active site. The energy minimized ligands were docked in binding site of APN.All the compounds were built using hyperchem version 8 and subsequently minimized. The protein structure was prepared for docking using AutoDock Tool. Polar hydrogen was added and non-polar hydrogen was merged and finally Kollman united atom charge and atom type parameters were added to 1L5G for RGD products and 2DQM for NGR products. Grid map dimensions for 1L5G (18.6378 × 41.9200 × 43.1259) were set surrounding active site. The energy minimized ligands were docked in binding site of αChemistryKetoprofen were selected as NSAIDs and fmoc peptide synthesis strategy was used to make both the peptide-drug conjugates and peptide-spacer-drug conjugates. Therefore, fmoc-6-amino hexanoic acid was needed to make the peptide-spacer-drug conjugates . The synBiological activityRGD and NGR are promising peptide sequences for targeted drug delivery to tumor cells. They can carry their drug conjugated to their target tumor. Ketoprofen was attached to N-terminus of the tripeptides RGD and NGR through its carboxylic acid group and thus an amide bond is responsible for the conjugation. In order to avoid the possible steric hindrance which is caused by the closeness of ketoprofen to the tripeptide, another from of the conjugate was also synthesized in which ketoprofen was attached to the tripeptide through a linear six carbon linker. Anti-proliferation activities of the synthesized compound were evaluated on a selected group of cancer cell lines. The results are summarized in a. No inhibitory activity was observed on MCF-7 (as a tumor cell without over-expressed RGD- or NGR-binding protein on its surface) or Fibroblasts .vβ3) on their surface. In case of A2780, keto-spacer-RGD showed higher inhibitory activity than keto-RGD (63 vs. 55% inhibition) which indicates possible role of spacer either in better binding to the receptor or in mitigating the steric hindrance for better binding of RGD to the cell surface. Surprisingly, the opposite pattern is observed in case of OVCAR-3 which shows the possible impediment of linker group for the ligand-receptor binding. b. ketoprofen caused no inhibition at 100 µm concentration either on A2780 or on OVCAR-3. However the conjugated forms of ketoprofen with RGD either with or without spacer, showed >50% inhibition on both cell lines. The two cell lines are well recognized for the overexpression of RGD receptors (A2780 with low and OVCAR-3 with high expression of αc. NGR conjugates of ketoprofen caused 3-7 times higher inhibition compared to ketoprofen on HT-1080 and SKOV-3. These are the cancer cell lines with over expression of NGR receptor (SKOV-3 with low and HT-1080 with high expression of CD13) on their surface. Therefore it could be speculated that the NGR companionship with ketoprofen has improved its cytotoxic activity on these cell lines. In both cases, the conjugated form which contains the spacer between ketoprofen and NGR showed higher activity which could be assigned to the less hindrance for ligand- receptor binding.Molecular modeling (docking) studiesAminopeptidase N (APN/CD13) which is assumed to be the target of ketoprofen-NGR conjugate is a zinc-dependent metalloprotease which is overexpressed in many disease such as cancer and inflammation. Bestatin is the first marketed APN inhibitor which has been introduced in 1976 (29). Therefor a docking study was conducted to investigate the possible binding mode of ketoprofen-spacer-NGR which has highest activity against HT-1080 cells. The result is presented in vβ3 integrin subunit that is overexpressed in cancer. In order to explore the possible mode of interaction for keto-RGD conjugate, docking study was carried out using 1L5G crystallography of αvβ3 integrin and the results are presented in 2 of asparagin moiety. The distance of Mn2+ ion in active site of enzyme from the carbonyl group of ketoprofen is 6.6A˚. Another Mn2+ ion in active site has 5.45A˚ distance from the carbonyl group of RGD peptide chain. Hydrophobic residue of (β)-Ala 218 with the distance of 3.9A˚ and 3.7A˚ has come into contact with ketoprofen rings. Hydroxyl group of (α)-Tyr178 forms hydrogen bond with NH of guanidine in keto-RGD with the distance of 3.88A˚ and also tyrosin ring has 3.49A˚ distance from CH2 side chain of Arg in keto-RGD.RGD target αRGD and NGR peptide sequences are capable of guiding a chemotherapeutic agent to its target. This strategy could be used for maximizing the efficiency of chemotherapy based on the fact that in many cancer cells, specific membrane receptors are overexpressed on the cell surface. The result of present study show that ketoprofen conjugated to RGD and NGR have higher cytotoxic activity compared to ketoprofen itself which strongly supports the hypothesis of targeting by peptide-drug conjugates.


The extent of genetic and epigenetic diversity between and within patient tumors is being mapped in ever more detail. It is clear that cancer is an evolutionary process in which tumor cell intrinsic and extrinsic forces shape clonal selection. The pre-clinical oncology pipeline uses model systems of human cancer – including mouse models, cell lines, patient-derived organoids and patient-derived xenografts – to study tumor biology and assess the efficacy of putative therapeutic agents. Model systems cannot completely replicate the environment of human tumors and, even within the same cancer model, data are often irreproducible between laboratories. One hypothesis is that ongoing evolutionary processes remain relevant in laboratory models, leading to divergence over time. In a recent edition of Nature, Ben-David and colleagues showed that different stocks of widely used cancer cell lines – a staple of cancer research over many decades – are highly heterogeneous in terms of their genetics, transcriptomics and responses to therapies. The authors find compelling evidence of positive selection based on ongoing mutational processes and chromosomal instability. Thus, the origin, culture conditions and cumulative number of population doublings of cell lines likely influence experimental outcomes. Here, we summarize the key findings of this important study and discuss the practical implications of this work for researchers using cell lines in the laboratory. Summary: A recent study in Nature demonstrated that ongoing mutational processes cause significant heterogeneity of widely used cancer cell lines. In this Editorial, the authors discuss the implications of these findings for the cancer and cell biology fields. To study human cancer biology, researchers can use patient biopsies or tissue from the surgical resection of tumors. However, this tissue represents a single snapshot in the life history of that tumor, typically at an advanced stage. In order to investigate the dynamic genetic and epigenetic course of cancer initiation, progression and metastasis, experimental model systems are required. More recent systems include genetically engineered mouse models (GEMMs) , patientin vivo, but are limited by species differences in oncogenic pathogenesis, the shorter lifespan of mice and, often, by the artificial, simultaneous introduction of multiple oncogenic driver events. Cell lines have the advantage of being derived from patients and are more easily manipulated in the laboratory. Nevertheless, the cancer research community has long appreciated that cell lines do not completely recapitulate human disease  were present in one data set but not the other. This discordance prompted the authors to examine the genetic variability within the cell lines in detail.https://portals.broadinstitute.org/ccle) and Connectivity Map (https://clue.io/cmap)]. The MCF7 strains also demonstrated substantial heterogeneity in their mutational status, chromosomal rearrangements and CNAs. Deep, targeted sequencing of 447 genes revealed that just 35% of coding non-synonymous single-nucleotide variants (SNVs) and insertions and deletions were shared by all of the strains, while 29% were unique to a single substrain. Even when specific mutations were present in all strains, the allelic fraction, defined as the number of variant reads divided by the total number of reads at a specific genomic locus, varied substantially. The authors highlight an activating PIK3CA mutation (G1633A), for which the allelic fraction fluctuated between 0.21 and 0.7, as an example likely to have functional consequences. Differences in larger genomic rearrangements, such as arm-level copy number events, and differential copy number gains and/or losses in hundreds of genes were also evident among strains. Of those events, only 13% of gains and 21% of losses were present across all strains, with 7% of gains and 13% of losses being unique to a single strain. Importantly, these events affected genes such as TP53, EGFR and PTEN, which are known to be gained or lost during breast cancer pathogenesis. This indicated that individual existing strains of the MCF7 cell line might be more biologically relevant to the study of estrogen receptor-positive breast cancer than others.Laboratories around the world maintain different substrains of cell lines, so the authors sourced 27 independent vials of MCF7, an estrogen receptor-positive breast cancer cell line, predominantly from two major Broad Institute projects [Cancer Cell Line Encyclopedia https://portals.broadinstitute.org/ccle) or the Sanger Institute's Cell Lines Project (https://cancer.sanger.ac.uk/cell_lines)] should be used for broad direction but specific mutation/pathway status should be confirmed locally.For existing cell lines, each laboratory's strain should be profiled. Reference genomes for cell lines [e.g. the Broad Institute's Cancer Cell Line Encyclopedia ((2)On adoption of new cell lines, identical cryovials of very early passage cell lines should be established on arrival and later passage working stocks derived from just one of these. Master stocks could then be used for validation of key findings prior to publication.(3)More rigorous monitoring and reporting of population doublings should become the norm, and substitute the current ‘passage number’ convention, which is imprecise. Cell lines should preferably be used within a defined window to minimize genetic drift.(4)The optimization, agreement and implementation of international ‘standard’ culture conditions for individual cell lines together with the documentation of heterogeneity metrics (e.g. measures of genomic divergence per passage in those conditions) in datasheets.(5)The reassessment of genomic landscape after significant culture bottlenecks induced by selection and/or single cell cloning (e.g. in CRISPR-Cas9 gene editing experiments).We believe that these data signal an end to the era in which researchers informally acknowledge that different strains of cancer cell lines are heterogeneous and unstable over long-term passage, but in practice treat them as though they were clonal entities. There are already established measures to minimize irreproducibility between laboratories, such as cell line identification , mycoplain vitro or in vivo, presents opportunities for researchers to improve the reporting and reproducibility of pre-clinical cancer research.In summary, Ben-David et al. reveal the scale of heterogeneity between strains of cancer cell lines and the ongoing nature of the processes that underlie this heterogeneity. Importantly, these processes are unlikely to be similar to the ongoing genomic processes in human tumors, and mean that as cell lines – already an idiosyncratic representation of a single patient's cancer – are expanded long-term, they become even less representative of human disease. Importantly, recognizing the variability within laboratory cancer models, whether


Picornaviridae, have been shown to have anticancer activity. Picornaviruses are small icosahedral non-enveloped, positive-sense, single-stranded RNA viruses infecting a wide range of hosts. They possess several advantages for development for cancer therapy: Their genomes do not integrate into host chromosomes, do not encode oncogenes, and are easily manipulated as cDNA. This review focuses on the picornaviruses investigated for anticancer potential and the mechanisms that underpin this specificity.Oncolytic viruses (OVs) form a group of novel anticancer therapeutic agents which selectively infect and lyse cancer cells. Members of several viral families, including  Herpes simplex virus-1 has shown increased efficacy when combined with cisplatin [Cancer remains one of the leading causes of death worldwide. Globally, there were around 18 million new cases of cancer in 2018, with up to 9.5 million deaths [isplatin . Oncolytisplatin ,5. Theseisplatin , Adenoviisplatin  and Mumpisplatin , and theHerpes simplex virus-1, also known as Talimogene Laherparepvec (T-VEC), by the US Food and Drug Administration (FDA) for the treatment of malignant melanomas [This was the approval of the first oncolytic virus, modified elanomas . In addielanomas ,11. Whilelanomas . This iselanomas . They alelanomas . ®), Vaccinia virus engineered to express GM-CSF with thymidine kinase deleted (PexaVec®), Measles virus engineered to express thyroidal sodium iodide importer (MV-NIS®) and naturally occurring Reovirus (Reolysin®). These viruses vary greatly with respect to several characteristics, including the species, genera, etc. to which they belong, their progress through clinical trials, cancer types they are used to treat and degree to which they have been engineered. Despite the inherent challenges presented in producing safe and effective oncolytic viruses, there are a number of naturally occurring and engineered viruses that have entered clinical trials. Included in their ranks are Adenovirus engineered to express granulocyte macrophage colony-stimulating factor (GM-CSF) , registered as CAVATAK . The onc3 TCID50 CVA21 (TCID—Tissue culture Infective Dose) and remained tumour-free until the end of the trial at day 35 [Potential oncolysis of a panel of six melanoma cell lines was inferred by increased levels of ICAM-1 and DAF . This wat day 35 . CVA21 wt day 35 ,39. Recet day 35 . Anothert day 35 . This reHigh ICAM-1 and DAF expression was confirmed in three multiple myeloma (MM) cell lines, with lower expression on peripheral mononuclear blood cells (PMBCs). MM cell lines showed cytopathic effect (CPE) on infection with CVA21 at a MOI as low as 0.2, increasing viral tires 100 to 1000-fold after 24 h. By contrast, CVA21 infection of PMBCs showed only modest CPE at MOI of 20 . When tr7 TCID50 CVA21 with a decrease in treatment tumour mass of 17 ± 6% compared to an increase in tumour mass of 2422 ± 932% in controls 26 days post-intervention [CVA21 was investigated for oncolytic activity against breast cancer . Of ninervention . CVA21 wrvention .To evaluate the effect of CVA21 on bladder cancers, a panel of ten bladder cancer cell lines were classified based on susceptibility . Six celThe combination of CVA21 with four common chemotherapy agents was assessed on a panel including breast cancer, colorectal cancer, pancreatic cancer and healthy cell lines . These eIn addition to native virus, the RNA of CVA21 transcribed from plasmid DNA has been shown to be cytotoxic on cell lines including hepatocellular carcinoma, lung cancer, rhabdomyosarcoma, and melanoma . This wa7, 108 or 109 TCID50 CAVATAK were safe, and five out of nine patients showed transient or stable tumour mass reduction, or stabilisation of tumours. In a Phase II clinical trial of CAVATAK in late stage melanoma (CALM), 57 patients were treated with up to 3 × 108 TCID50 CAVATAK intratumourally on days 1, 3, 5, 8 and 22, followed by injections once every three weeks for 18 weeks. This treatment regime resulted in no NCI CTCAE  grade 3 or 4 events, with 38.6% of patients showing immune-related progression-free survival at six months, and 19.3% of patients showing durable objective responses [The oncolytic activity of CVA21 in a diverse range of cancers both in vitro and in vivo prompted several clinical trials . A Phaseesponses . In an eesponses .8 TCID50), medium  and high  [In a Phase I/II clinical trial on systemic treatment of resistant malignancies (STORM), the enrolled patients with late-stage cancer were grouped into three CAVATAK dose cohorts, low  ,47. The  TCID50) .8 TCID50, 3 × 108 TCID50, or one of each. The second part of the study tested the combination therapy of two doses of 3 × 108 TCID50 CAVATAK with 10 mg MC. The study established the safety of CAVATAK and MC and recorded viral replication within tumours, complete tumour response and induced inflammation and apoptotic cell death [Revisiting the study of CVA21 with mitomycin C (MC) against bladder cancer cell lines, a Phase I/II clinical trial of CAVATAK in non-invasive bladder cancers (CANON study) was undertaken . The firll death .CVA21/CAVATAK as an oncolytic agent has garnered the greatest amount of research among the coxsackieviruses. However, the infection of CVA21 in humans is common, and pre-existing adaptive immunity to this strain could result in premature clearing of the virus before being able to exert its therapeutic effect on tumours. This prompted an evaluation of alternatives to CVA21 with less prevalent rates of immunity.Coxsackievirus serotypes A13, A15 and A18  have all been shown to infect and productively replicate in multiple melanoma cell lines, while not infecting peripheral blood mononuclear cells (PMBCs) . A studyOf 28 enteroviral strains tested against 12 human cancer cell lines and a bone marrow stroma control cell line, Coxsackievirus strains B2 Ohio-1 (CVB2 Ohio-1), B3 Nancy (CVB3 Nancy) and B4 JBV (CVB4 JBV) stood out as causing cell death in A549 and LK-87 lung cancer cells with no toxicity in controls . In a seN- and 6-O-sulfated heparan sulfate (HS) as a cellular receptor [Four strains of Coxsackievirus B3  were tested against colorectal cell lines expressing varying levels of CAR and DAF . Of the receptor . HS is areceptor . CVB3 Nareceptor .Three endometrial cancer (EC) cell lines, Ishikawa, HEC-1-A and HEC-1-B, were shown to express the cellular receptors for CVB3. When infected by CVB3 2035A at an MOI of 10, there was extensive lytic destruction of all three cell lines and high titres of viral progeny produced after 48 h . Mouse sFinally, Coxsackievirus B6 (CVB6) in the form of three non-pathogenic live enterovirus vaccine strains  was tested against healthy and cancer cell lines. LEV15 stood out as having very little infectivity in untransformed cell lines and varying infectivity in the cancer cell lines tested . LEV15 wPoliovirus is the causative agent of poliomyelitis, a disease infamous for causing life-long disability and paralysis throughout human history. Infection with Poliovirus (PV) is usually asymptomatic or manifested as transient flu-like symptoms and/or gastroenteritis . An esti6 TCID50 LAPV were sufficient to control tumour growth by 40% and induced cancer cells apoptosis, as shown by terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) assay [Live Attenuated Poliovirus A vaccine containing the Sabin 1 strain (LAPV) has been tested for oncolytic activity in six human bone and soft tissue cancer cell lines expressing moderate to high levels of CD155, as well as one murine osteosarcoma . All butL) assay .Vaccinia virus vector that provides the P1 region [One way to attenuate PV neurotoxicity is by using replication incompetent Poliovirus 1 (PV1) replicons. Replicons are constructed by deleting the P1 region that encodes the capsid proteins . Once in1 region . PV1 rep1 region .133Gmono-cre illustrates another strategy to neuro-attenuate oncolytic PV [133G, was present in both cases, and so A133Gmono-CRE PV was isolated and used in further studies. Along with pre-exposure to mono-CRE PV, four intratumoural injections of A133Gmono-CRE PV into neuro-2a xenograft tumours were sufficient to clear tumours in 82% of mice, and also effectively vaccinate against re-challenge with neuro-2a cells [133Gmono-CRE PV cultures also achieved this effect [The PV mutant Alytic PV . Initiallytic PV . The ratlytic PV . In vitr2a cells . CD8+ T-s effect . These rBy far the most promising prospect for the use of PV in a cancer therapeutic setting is the chimeric virus, PVSRIPO. PVSRIPO ”) is the Sabin strain of PV1 (PVS) with IRES replaced with that of human Rhinovirus 2 (HRV2) ,82. The The basis for neuronal attenuation of PVSRIPO is multifactorial. Wild-type polioviral IRES binds the eIF4G:4A:4B complex (eukaryotic initiation factor 4G:4A:4B), unwinding the RNA, exposing the start codon and binding eIF3, while the HRV2 IRES does not in neuron lineage cells . PVSRIPOPVSRIPO has demonstrated potent oncolytic activity in a wide range of cancer cell lines derived from gliomas , breast Infection of ex vivo glioma primary cultures derived from stage II–IV patients with PVSRIPO showed drastic CPE by 6 h and complete CPE by 12 h, resulting in viral titres comparable to those produced by established glioma cell lines . The ex 7 plaque formation units (PFU) or a high dose of 109 PFU of PVSRIPO. In the intracranial model, the distinction between the administered doses of PVSRIPO was more pronounced, with the low dose extending median survival times by 122% and the high dose increasing median survival times by 153% compared to controls [CD155 expression in human breast cancer cells is relatively low . Howevercontrols .7 PFU) or high dose (109 PFU) PVSRIPO. While there was no significant dose response relationship between the two dose cohorts, median survival compared to controls increased by 187.5% and 200%, respectively, with one of ten mice in the low dose cohort, and three of ten mice in the high dose cohort surviving to the end of the trial [8 TCID50 PVSRIPO in both tumours showed an average of a 45% decrease in tumour mass by day 10, nearly clearing the xenografts by day 28. Tumours isolated from mice sacrificed at day 10 and 28 showed a temporal progression towards tumour death, with day 10 tumours displaying a dense core of intact cancer cells surrounded by a loose mantle of dispersed tumour cell foci and immune infiltrates. At day 28, the tumours were composed primarily of scar tissue, with minor foci of heterogenous composition [PVSRIPO was also tested in a glioblastoma mouse model . Athymiche trial . The acthe trial . Athymicposition . PVSRIPOposition .In melanoma cells, PVSRIPO effectively evades the innate immune response, where other related viruses such as Enchephalomyocarditis virus (EMCV) cannot, and the mechanism of this is independent of melanoma differentiation-associated protein 5 (MDA5) and the mitochondrial antiviral signalling protein (MAVS) .In vitro studies with SUM149 breast cancer cells and DU145 prostate cancer cells showed PVSRIPO had potent activity against these cell lines and that their infection released pro-inflammatory cytokines . By day PVSRIPO has been shown to infect human THP1 macrophages, but unlike many of the cancer cells studied, PVSRIPO infection of macrophages is sublethal and serves to induce expression of major histocompatibility complex class II and costimulatory molecules and further leads to (interferon-β) IFN-β, (interleukin-12) IL-12 and (tumor necrosis factor- α)TNF-α production . The imm7, 109 or 5 × 109 TCID5 PVSRIPO showed no side-effects, and all survived to the end of the trial [7 TCID50 [Following the recommendations of the World Health Organisation (WHO) for putative Poliovirus vaccines, before starting human trials, PVSRIPO had to be tested in primates. Ex vivo cultures of macaque and human kidney cells showed no species-specific infectivity for either PVS or PVSRIPO . Macaquehe trial . The virhe trial . These rhe trial ,94. A Ph7 TCID50 . In addi7 TCID50 .Enteric cytopathic human orphan (ECHO) viruses are part of the species Enterovirus B. Several echoviruses have been evaluated for selective tropism towards different cancers, including EV7, EV1, EV5, EV12, EV15, EV17, EV26 and EV29.Most abundant information regarding clinical applications is available on Echovirus-7 (EV7), registered by the name Rigvir . Rigvir Rigvir has shown toxicity in vitro against a broad range of cell lines, including melanoma, pancreatic adenocarcinoma, muscle rhabdomyosarcoma, mesenchymal stem cells, gastric carcinoma, lung carcinoma and human normal dermal fibroblasts .Clinical trials are referred to in the literature to have occurred between 1965 and 1991, but the results from these clinical trials are not readily available [One such retrospective study showed a statistically significant increase in 3-year survival of melanoma patients when treated with Rigvir post-surgery than surgery alone or with other immunomodulators . For besIn recent times, case studies of Rigvir treatment in a diverse range of cancers have been emerging in the literature. One such study followed a stage IV melanoma, stage IIIA small-cell lung cancer and stage IV hystiocytic sarcoma on a long-term Rigvir treatment . FollowiRigvir as a monotherapy and in combination with other therapies has also been tested in patients for whom the prognosis is especially poor. One patient with a basal cell carcinoma, a diagnosis with a median expected survival of five months, was treated with Rigvir post-surgery . The treα2β1, has been identified as the cellular receptor for EV1 [α2β1. This effect could be ablated by treatment with anti-α2β1 antibodies [Other Echovirus species have also been investigated for activity against cancer. Echovirus 1 (EV1) has been tested for activity against ovarian, prostate and gastric cancer cell lines. VLA-2, also known as integrin  for EV1 . Consisttibodies . EV1 wastibodies .α2β1, EV1 selectively destroyed the cancer cell lines [3 diameter was sufficient to reduce them to an average of 1 mm3 [3 and 107 TCID50 were used to test the efficacy of EV1 in large LNCaP tumours of 300 mm3. The low dose of EV1 had a transient but significant protective effect, while the mid- and high doses of virus showed lasting significant tumour suppression [While several prostate cancer cell lines as well as healthy prostate cell lines were also shown to express ll lines . Moreoveof 1 mm3 . Differepression .α2β1 was investigated [−6 to 3 × 10−2 TCID50/cell in MKN-45, AGS and NCI-N87 and >1 TCID50/cell in Hs746T. While Hs746T infection in vitro produced only minimal CPE and viral replication after 72 h, the other three cell lines produced a 100-fold increase in EV1 viral titre after 12 h [3 TCID50) responsible for a 47.38% reduction, the medium dose (105 TCID50) for a 68.5% reduction and a 71.5% reduction in the high dose (107 TCID50) cohort. For reference, mice treated with PBS had an average flux increase of 28.75%, and most were sacrificed due to disease progression. Interestingly, circulating virus was initially different across dose groups, but then equalised [The minimum infectious dose of EV1 to produce CPE in four gastric cancer cell lines expressing stigated . These wter 12 h . MKN-45 qualised .Echovirus 5 (EV5) represents an interesting study into the oncolytic activity of the wild-type virus genome and the naked RNA transcribed from a cDNA clone . EV5 wasFinally, the broader oncolytic ability of Echoviruses was tested in a study with viruses including EV12, EV15, EV17, EV26, and EV29 against a selection of six colorectal cell lines . EV12 atBovine Enteroviruses are endemic in cattle, being routinely isolated from cow faeces, and are not pathogenic to humans . Bovine Vibrio cholera neuraminidase (VCN) showed a dose- and time-dependent inverse correlation between VCN exposure and viral titre following BEV 261 infection in a panel of susceptible cancer cells [BEV1 was first investigated for antitumour activity in 1971, wherein 65% of cancer cell lines met the threshold for significant viral killing by BEV1, compared to 4% of healthy cell lines . The perer cells .Interestingly, whereas other picornaviruses can bio-adapt to cell lines (see: Echovirus 7), this could not be achieved with BEV1 . The samIn addition to murine studies, BEV1 was also tested in rabbits and dogs and shown to be well tolerated . Mice beSeneca Valley Virus (SVV) was originally discovered as a contaminant of cell culture . The sel8 viral particles per kilogram (vp/kg) SVV could clear pre-established H446 xenograft tumours and most mice treated with 107 vp/kg cleared tumours, with two exceptions. Similarly, when athymic mice with Y79 retinoblastoma xenograft tumours were treated with 108, 1011 or 1014 vp/kg SVV, six of eight, seven of seven and five of seven mice, respectively, cleared the tumours [The oncolytic potential of SVV was first highlighted in a 2007 study . Screeni tumours . This tr13 vp/kg SVV [Concurrently, SVV was shown to have activity against Weri retinoblastoma cells in vitro . Doses ap/kg SVV .12 vp/kg. SVV treatment increased the survival times in mice of both tumour sizes. Of note is that SVV could cross the blood–brain barrier (BBB) and did not infect any healthy mouse brain cells [To research SVV in the application against medulloblastoma (MB), mouse MB models were established using ex vivo primary cultures isolated from 10 paediatric patients . Primaryin cells .50  values of 1.6 × 10−3 vp/cell, 3.1 × 10−4 vp/cell and 3.9 × 10−3 vp/cell, respectively. Gene expression profiles of permissive and refractory cell lines showed that higher expression of late neurogenic transcription factor NEUROD1 and lower expression of the early neurogenic transcription factor ASCL1 correlated with permissivity to SVV [9 to 1011 vp/kg SVV, and two out of six mice completely cleared tumours when treated with 1014 vp/kg SVV. Kinetics studies showed peak replication of SVV occurred at day three [Small-cell lung cancer (SCLC) is an aggressive type of tumour. There are two SCLC groups: classic and variant, which make up for 70% and 30%, respectively [Since SVV could exert a therapeutic effect in medulloblastoma models, it raised the question of its applicability in other cancers for which the BBB presents a significant obstacle, such as gliomas. In a study of six ex vivo glioma primary cultures, four were found permissive to SVV infection at an MOI as low as 0.5, while the other two remained resistant up to an MOI of 25 . Replica12 vp/kg NTX-010 were seen in neuroblastoma, rhabdomyosarcoma, rhabdoid tumour, Wilms tumour and glioblastoma cell lines [SVV-001  is registered by Neotropix inc. as NTX-010 . A preclll lines . Taken t7 to 1011 vp/kg showed no dose-limiting toxicities, with flu-like symptoms mainly manifesting in the lower dose cohorts. Neutralising antibodies were detected as early as two weeks into treatment. In terms of outcomes, one patient showed disease stabilisation, while another five had minor responses, which were not sufficient to meet RECIST criteria  [9, 1010 or 1011 vp/kg NTX-010, and in part B, patients were given oral (days 1 to 14) and intravenous (days 1 and 8) cyclophosphamide, in combination with two doses of 1011 vp/kg NTX-010. The study showed that NTX-010 was well tolerated with a single dose-limiting toxicity event recorded. While no objective response was observed, six patients did show disease stabilisation. A rapid neutralising antibody response to NTX-010 was also shown in this trial [11 vp/kg NTX-010, or Arm B, the saline control. Grade 4 adverse events were seen in three of the Arm A patients and none of the Arm B. Between the two cohorts, there was no difference in progression-free survival, and overall survival was slightly less in Arm A (83%) vs. Arm B (85%). The trial was also prematurely terminated to investigate the death of one patient, which was confirmed to be unrelated to NTX-010 treatment [First, a phase I clinical trial of 30 patients with SCLC was undertaken . Patienttumours) . The pattumours) . Shortlytumours) . In partis trial . Finallyis trial . A cohorreatment . It is ireatment . Based oTheiler’s murine encephalomyelitis virus (TMEV) is a natural murine pathogen that has been engineered to express tumour antigens to induce a targeted antitumour immune response and thus act as a cancer vaccine.TMEV engineered to express chicken ovalbumin (TMEV–OVA) can generate host immune responses against OVA-expressing tumours ,120. The121–130) [121–130 region has been attempted in order to direct a greater proportion of the cytotoxic immune response to expressed foreign antigen. Deletion of this immunogenic region is shown to increase the relative amount of CTLs primed against foreign antigen [During the host immune response generated to TMEV expressing foreign antigen, the adaptive immune response targets a combination of the foreign antigen and TMEV antigens. TMEV is known to harbour a highly immunogenic region in the VP2 structural protein (VP2121–130) . Suppres antigen . Vector  antigen . However antigen . In fact antigen .Wild-type DA strain of TMEV has been investigated in vivo against breast cancer and melanoma cell tumours, without a significant protective effect in either . Wild-tyEncephalomyocarditis virus (EMCV) was discovered in 1945 when a captive male gibbon suddenly and inexplicably died of pulmonary oedema and myocarditis . Mice trAs far back as 1965, EMCV, referred to as Columbia-SK virus, showed oncolytic activity against fructose sarcomas in mice and rats, with a more pronounced effect in rats . The intVHL is a tumour suppressor protein with a role in tumour necrosis factor (TNF)-α mediated cell apoptosis  and oxyg24, a poly-C truncated attenuated MEV, demonstrated oncolytic activity in a miscellaneous panel of cancer cell lines that included several multiple myeloma cells of human and murine origin [24 caused significant CPE in the majority of cancer cell lines tested [24 infection. BALB/c mice bearing MPC-11 tumours were treated with either a single intravenous (IV) injection of 107 TCID50 vMC24 or intratumoural (IT) injection of 106 TCID50 vMC24. While IV injection showed only minimal tumour suppression, IT administration of vMC24 resulted in a decreased tumour volume within five days. However, three mice in the IV group and two in the IT group developed paralysis, with thee more dying overnight, suggestive of a heart failure [24 showed potential as oncolytic agent but with lethal toxicity. This prompted a search for novel strategies to attenuate vMC24, such as the incorporation into the viral genome of sequences complementary to healthy tissue specific micro-RNAs [24 variants with complementary micro-RNA sequences (miRT viruses) were generated. The miRT viruses had combinations of miR125b (expressed in brain tissue), miR124 (expressed in neurons), miR133 and miR208a (enriched in cardiomyoctyes). Among these, a construct labelled vMC24-NC stood out as having very low viral titres in the brain, spine and heart of experimentally infected C57BL/6 mice. vMC24-NC had two miR124 sequences in the 5′ UTR, and one miR133b and one miR208a in the 3′ UTR. In vivo experiments with mice bearing MPC-11 tumours showed that four of ten mice treated with vMC24-NC cleared tumours and two delayed tumour growth, with no negative side-effects. The mice treated with vMC24 had a rapid tumour regression, but seven out of ten developed paralysis. Furthermore, vMC24-NC was also shown to eliminate tumours in a dose-dependent manner while remaining genetically stable in vivo [Similar to Poliovirus, the research into oncolytic Mengovirus (MEV) focused around novel attenuation methods . A studye origin . vMC24 cs tested . Of the  failure . Therefocro-RNAs ,128. Mul in vivo .This review discussed picornavirus species investigated for anticancer activity . These v


Additionally, high extracellular lactate cultures were labelled in parallel with [U-13C] lactate, which provided qualitative information regarding the lactate uptake and metabolism. The 13C-MFA model, which incorporated the measured extracellular fluxes and the parallel labeling mass isotopomer distributions (MIDs) for five glycolysis, four tricarboxylic acid cycle (TCA), and three intracellular amino acid metabolites, predicted lower glycolysis fluxes in the high lactate cultures. All three cell lines experienced reductive carboxylation of glutamine to citrate in the TCA cycle as a result of high extracellular lactate. Reductive carboxylation previously has been observed under hypoxia and other mitochondrial stresses, whereas these cultures were grown aerobically. In addition, this is the first study to investigate the intracellular metabolic responses of different stages of breast cancer progression to high lactate exposure. These results provide insight into the role lactate accumulation has on metabolic reaction distributions in the different disease cell types while the cells are still proliferating in lactate concentrations that do not significantly decrease exponential growth rates.In cancer tumors, lactate accumulation was initially attributed to high glucose consumption associated with the Warburg Effect. Now it is evident that lactate can also serve as an energy source in cancer cell metabolism. Additionally, lactate has been shown to promote metastasis, generate gene expression patterns in cancer cells consistent with “cancer stem cell” phenotypes, and result in treatment resistant tumors. Therefore, the goal of this work was to quantify the impact of lactate on metabolism in three breast cell lines , in order to better understand the role lactate may have in different disease cell types. Parallel labeling metabolic flux analysis ( For example, when lactate was injected into mice with xenografts of the human breast cancer cell line MDA-MB-231, metastasis increased ten-fold  glucose, [U-13C] L-glutamine, and [U-13C] sodium lactate, where the control cultures were only labeled with  glucose and [U-13C] L-glutamine. The lactate labeling data for the high-lactate cultures provided uptake and direct intermediate tricarboxylic acid (TCA) cycle labeling information. The intracellular metabolic fluxes were predicted for each cell line and each condition from the glucose and glutamine labeling data.The aim of this study was to determine the role of extracellular lactate on the metabolism of three different proliferating breast cell lines . Each cell line was grown in a low glucose control  media or in a high-lactate media, where sodium lactate was added to the control medium to make the high-lactate medium. The high-lactate concentration was selected for each cell line such that equivalent exponential growth rates for the control and high-lactate cultures were maintained. These equivalent exponential growth rates were important for the modeling assumptions for two reasons: 1) the quasi-steady state assumption was met equally well by both conditions, and 2) it allowed for normalizations of fluxes between the conditions for the same cell line based on biomass generation. Parallel labeling experiments were conducted with  glucose, [U-13C] L-glutamine, and [U-13C] sodium lactate, all purchased from Sigma-Aldrich.MCF 10A (ATCC CRL-10317), MCF7 (ATCC HTB-22) and MDA-MB-231 (ATCC HTB-26) cells were from the American Tissue Culture Collection (ATCC). MCF 10A cells are a non-tumorigenic breast cell line, MCF7 cells are a tumorigenic, luminal breast cancer cell line, and MDA-MB-231 cells are a metastatic, basal breast cancer cell line . Dulbecc4 cells/cm2 into either 6-well plates or T-25 flasks in the control growth media. Cells were cultured at 37°C in a 5% CO2 humidified incubator. After 24-h, the media for the MDA-MB-231 cultures was exchanged and the experimental conditions were introduced. The media exchange time is used to set time 0 for all cell lines. For MCF 10A and MCF7 cell lines the lag phase was longer, so the media was replenished at 24-h post seeding. The media was exchanged 48-h post seeding for MCF10A and MCF 7 to introduce the experimental conditions. For clarity, the full experimental setup is shown in 13C] glucose) and glutamine tracers (95% molar enriched [U-13C] L-glutamine) were performed for the control cultures, whereas the high-lactate cultures also had parallel replicates for the lactate tracer (50% molar enriched [U-13C] sodium L-lactate). Six-well plates (Nunc) were used to obtain cell numbers and glucose and lactate concentrations with six replicates for each time point. T-25 flasks were used to obtain extracellular amino acid concentrations and intracellular MID measurements in triplicate.All cell lines were seeded at 2 x 10Cell numbers were obtained using the Scepter 2.0 Handheld Automated Cell Counter (Millipore). Glucose and lactate concentrations were measured using a YSI 2700 Bioanalyzer.13C] algal amino acid solution, which were then used to quantify the amino acid concentrations in the extracellular medium.Intracellular and extracellular metabolites and specific metabolite fragments used for identification were extracted and derivatized according to the protocol outlined by Ahn and Antoniewicz , with a GC-MS analysis was performed using a Hewlett Packard 7683 GC equipped with an HP-5  capillary column, interfaced with a Hewlett Packard 5973 MS operating under ionization by electron impact as 70 eV and 200°C ion source temperature. The injection port and interface temperatures were both 250°C while helium flow was maintained at 1 mL/min. Mass spectra were recorded in full scan mode for amino acid quantification and in single ion mode (SIM) for MIDs as well as internal standards and standardization curves. MIDs were obtained by integration of single ion chromatograms and corrected for natural isotope abundances using the Metran software , 32.-1  glucose included in the MFA simulations were 3-phosphoglycerate (3PG), dihydroxyacetone phosphate (DHAP), pyruvate, lactate, and alanine. The MIDs metabolites labeled by [U-13C] glutamine included in the MFA simulations were succinate, malate, α-ketoglutarate (AKG), glutamate, citrate, glutamine, and pyruvate. The MIDs of metabolites labeled by [U-13C] lactate were not included in the MFA simulations.e Metran , which ue Metran . Metaboltal data , 36. In tal data , 21. Mosμ is the growth rate (h−1). C48 and C24 are the individual metabolite concentrations (mM) at 48 and 24 hours after the media exchange, respectively. X24 is the cell concentration (cells/mL) at 24 hours after the media exchange. In Meadows et al. (2008)  glutamine) pathways [13C] glucose, the percent isotope labeling for DHAP, 3PG, pyruvate, alanine, and lactate were 40% or higher for all three cell lines. No significant differences were observed for MIDs from glycolytic metabolites  between the three cell lines and between the control and high-lactate conditions. These results reflect the consistently high flux through glycolysis for each of the three cell lines and the equivalent growth rates between conditions within a given cell line. These results also confirm that the 24-h labeling time was sufficient to reach isotopic steady state for  glucose. The intracellular MID profiles for all three cell lines are included in Fig B in  glutamine. Using [U-13C] glutamine, the observed isotopomer labeling for intracellular glutamine, AKG and glutamate was over 70%. 13C] glutamine, as an example, for the TCA metabolites. Pyruvate and lactate were minimally enriched from the glutamine isotope across the cell lines and both conditions, where For the three cell lines and both conditions, glutamine, AKG, and glutamate were highly labeled from [U-13C] glutamine tracer. The MID profiles for these metabolites were similar for all three cell lines; however, citrate MIDs were significantly different between the control and high-lactate cultures within a cell line, indicating the effect of lactate was significant. More specifically, a significant increase in M5 citrate labeling was observed in the high-lactate conditions for all three cell lines Click here for additional data file.