Inside the absence of full and accurate experimental information, computational methods are becoming a vital tool for probing the interactions of integrase with inhibitors and substrates. Fragmented information concerning the framework of HIV-1 IN happen to be utilised to construct models to enhance our comprehending of inhibitor binding to the target. Theoretical versions of both the dimer and tetramer states have already been constructed. De Luca and coworkers described a dimeric model on the full-length IN/viral DNA complicated with two Mg2+ cations while in the active internet site, steady with cross-linking data indicating the Q148 and Y143 residues interact with viral DNA . The molecular docking strategy has also been employed to investigate even further the interactions of the HIV-1 IN dimer with viral DNA before the 3′ processing reaction . Most theoretical models give some thought to a tetrameric IN alone or in complicated with both viral DNA or viral DNA/ target DNA .
The influence of metal ions on IN?DNA complexes continues to be explored within a tetramer model constructed by homology modeling and MD simulations . It had been noticed that metal cations could mTOR inhibitor potentially influence the location within the viral DNA on IN. Full-length designs of your HIV-1 IN tetramer in complicated with both viral and target DNAs are actually constructed with both 1 or two Mg2+ ions during the active web site, to make certain consistency with biochemical experimental findings. The molecular docking of various DKAs onto the catalytic core domain identified two completely unique binding regions within the energetic web-site, including both the conserved D64- D116-E152 motif or the versatile loop area formed by amino acid residues 140-149, and confirmed that the mechanism of inhibition by DKAs calls for metal chelation by the ?-ketoenol group .
A comparative residue interaction examination was not too long ago performed , enabling evaluation of the non bonded interaction energies with the inhibitors with person energetic web-site residues and an evaluation from the correlation with biological action, major for the identification of crucial residues and characterization of interactions concerning the ligand and receptor. The designs recommend that Asp64, Thr66, Val77, Asp116, Glu152 and Lys159 are the critical residues influencing the binding of ligands with all the integrase. The docking of raltegravir and analogs onto Mg2+-complexed IN demonstrated the establishment of direct interactions involving raltegravir along with the three catalytic residues D64, D116, and E152, and with residues T66, E92, Y143, Q148, and N155 .
This outcome was once more constant using the findings of clinical experimental resistance profiling and presented a rational for the involvement of E92 and Y143residues in resistance. A single crystal framework of the IN core domain co-crystallized with an INSTI has become obtained with 5CITEP . The inhibitor is located among the energetic web site residues D64, D116 and E152 .

The class I PI3K results cellular functions by its two main downstream effectors Akt and mTOR. Akt can phosphorylate FoxO3a, BAX, Poor, and caspase 9 to antagonize apoptotic activity, phosphorylate prosurvival factors such as MDM2 and IKK-? to preserve cell survival, phosphorylate mitochondrial hexokinase- II to prevent mitochondria from initiation of apoptosis, phosphorylate GSK3 and cell cycle inhibitors p21WAF1 and p27KIP to promote G1/S cell cycle progression, phosphorylate tuberous sclerosis complex two or PRAS40 to trigger mTOR complex 1 – mediated protein synthesis, and phosphorylate telomerase reverse transcriptase to increase cell longevity . The mTOR kinase acts as an Akt substrate when mTOR binds to Raptor to formmTORC1. ButmTOR can grow to be an Akt upstream activator when mTOR binds to Rictor to form mTOR complicated 2 mTORC1 promotes protein synthesis by activation of its two downstream pathways: p70S6 kinase /S6 ribosomal protein pathway triggers translation of 5′ terminal oligopolypyrimidine mRNAs encoding ribosomal proteins and elongation components and eukaryotic translation initiation aspect 4E -binding protein 1 / eIF4E pathway initiates cap-dependent translation .
Accumulating proof exhibits that regulation of eIF4E activity can be a two-step mechanism. At first, active mTORC1/4EBP1 signaling leads to dissociation of eIF4E from 4EBP1 binding, which in turn allows Erk extra resources and/or p38 MAPK-mediated MnK1 and Mnk2 to phosphorylate eIF4E on ser209, consequently facilitating eIF4E to enter the eIF4F complicated and triggering cap-dependent translation . The cap-dependent translation can synthesize proteins selling cell growth and neovascularization and a few malignant behaviours connected to tumour progression .
It’s been reported that numerous molecular alterations in any element with the PI3K pathway more helpful hints and its upstream signals can result in constitutive activation of PI3K kinase cascades. This consists of mutations identified in genes encoding RTKs this kind of as mutant KIT-driven human and canine mast cell tumours and mutant Flt3-driven leukemia . Mutations of K-ras and N-ras genes have already been documented in canine lung cancer and canine leukemia respectively . Aberrant expression of class I PI3K subunits, this kind of as amplification of PIK3CA and mutation of PIK3R1, is normally uncovered in colon cancer . Substantial frequency of PTEN mutation has become reported in malignant glioblastoma . In addition, post-translational modification of PTEN, leading to down-regulation of PTEN action, has become described in T cell leukemia .
Alterations of 3 Akt isoforms, including amplification of Akt1, somatic mutations of Akt1,amplification of Akt2, overexpression of Akt2 with no evidence of Akt2 amplification, overexpression of Akt3 mRNA and protein but lack proof of Akt3 amplification, and somatic mutations of Akt3 are actually reported within a broad range of tumour kinds .

Fibronectin-1, which mediates cell and tissue cohesion, is also up-regulated in pancreatic along with other cancers . In other tumor cell versions, cellular anxiety induced MRP1 and P-gp overexpression leading to enhanced Gemcitabine sensitivity, which may very well be abolished by blocking these efflux pumps with verapramil . Enhanced chemoresistance in 3D culture Beside the molecules leading to greater chemoresistance, we had been also interested whether we could identify novel substances that will be capable of acting in 3D. A distinction in sensitivity to Gemcitabine, the common for pancreatic cancer treatment method, concerning 2D and 3D culture methods, as described previously , was verified in this research being a handle: in 2D cultures Gemcitabine reduced cell viability of BXPC3 and Capan- 1 to 40-60%, whereas Panc-1 cells were rather resistant to the remedy, and larger Gemcitabine concentrations had been required to affect cell viability .
As expected, PSC cells included as a non-transformed management cell line selleck Wnt signaling inhibitor have been quite possibly the most sensitive to treatment method each in 2D and 3D cultures . A panel of medicines with different targets was tested at two or 3 concentration ranges on the two 2D and 3D cultures . A lot of the compounds tested, such as the microtubule inhibitors CB5 and CB7, the anti-metabolites MT100, allicin, as well as flavonoid AXP decreased cell viability to 20-60% at the highest concentration in 2D culture. The result from the identical compounds on the 3D culture was much decrease and only some reduced cell viability maximal to 65% and around 40% . The mode of action and molecular mechanisms of those two compounds are subject of even more studies.
Testing medicines in the 3D culture model raises the concern of drug penetration, which may be impaired by structural features within the three dimensional culture, such as the dimension of the spheroids . Drug penetration into the spheroid is additionally established by diffusion with the ECM. The exact interactions concerning cancer cells and their microenvironment, both cell-cell and cell-matrix adhesion, are amongst the variables that ascertain the result of chemotherapy , and therefore are possible to differ from one particular cell type to a different. PDAC cells express by now endogenous ECM parts this kind of as collagen and fibronectin-1 . Higher drug resistance was shown in PDAC cells grown on fibronectin-1 or collagen coated culture dishes .
In our research the acquisition of elevated drug resistance of cancer cells within the 3D culture model may possibly be explained by the increased endogenous ECM protein expression within the microenvironment of your spheroids, so supporting the proposed cell adhesion-mediated drug resistance , and upregulation of other, alot more lately identified molecules described above, e.g.

To glean even further insight into the selectivity of these compounds for the distinct cell lines, we examined the relative expression of VEGFR2 and FGFR1 in HeLa, pHFF and HUVECs by executing immunoblotting on total cell lysates compared with known amounts of recombinant receptors . Only HUVECs express VEGFR2 protein . Having said that, all three cell lines express differing quantities of FGFR1: HeLa ??HUVEC ??pHFF . HUVECs express ~25% of VEGFR2 compared with FGFR1 . These information propose that inhibition of fibroblast wound closure by indolinones and anilinophthalazines just isn’t resulting from inhibition of VEGF-A regulated pathways, but doesn’t make clear why these compounds inhibit fibroblast rather than HeLa wound closure. On top of that, we subjected HUVECs to a development component gradient and permitted them to migrate across a pore-containing filter inside the presence or absence of inhibitor.
Beneath control ailments, VEGF-A and bFGF elicited the TAK-438 strongest migratory responses even though EGF elicited a significantly less pronounced response . The two indolinones and anilinophthalazines inhibited VEGF-Amediated migratory responses in HUVECs to differing extents, SU5416 owning the best inhibitory effect and PTK787 acquiring the least inhibitory impact . All three compounds inhibited bFGFmediated migration to your very same extent whereas they failed to significantly inhibit an EGF-mediated migratory response in HUVECs . Considering indolinones and anilinophthalazines inhibit signalling by the ERK1/2 pathway in HUVECs, we also examined drug inhibition of cellular proliferation. Using both an MTS-based cell proliferation assay in addition to a BrdU uptake ELISA, it had been proven that neither drug considerably inhibited cell proliferation during the concentration variety one nM to one mM .
Indolinones and anilinophthalazines inhibit endothelial tube formation while in the presence of the two VEGF-A and bFGF The potential of endothelial cells to type into threedimensional tubular structures is critical for lumen formation while in blood vessel sprouting . Right here we use an in vitro co-culture model through which endothelial cells in the presence of either VEGF-A or bFGF type Triciribine hollow tubes on major of a confluent fibroblast monolayer. In an effort to assess the effects of indolinones and anilinophthalazines on tube formation we measured each the tubule length plus the amount of tubule branch points. SU5416 was the weakest inhibitor of tube formation . At one hundred nM, SU5416 inhibited VEGF-A-stimulated tube length development by ~31% and tube branch formation by ~64%.
Yet, on the exact same concentration, SU5416 failed to substantially inhibit bFGF-mediated tubulogenesis. At a increased concentration, 1 mM, SU5416 inhibited bFGF-driven tube formation by 90% or extra . Sutent was the additional potent inhibitor of tube formation and showed virtually finish inhibition of VEGF-A- and bFGF-mediated events at either 100 nM or 1 mM .

One more naturally taking place fibrous mineral getting bodily properties similar to asbestos, is erionite. Even so, this asbestos-like mineral is just not regulated by the US Environmental Protection Company as an asbestos fibre. Nevertheless, erionite is known as a carcinogen and listed from the International Company for Investigate on Cancer being a group I carcinogen . It’s been shown that publicity to erionite benefits in pleural and interstitial fibrotic alterations, which are much like individuals observed with asbestos. On top of that, in vitro scientific studies demonstrated that erionite, but not asbestos, is sufficient to induce malignant transformation of cultured human mesothelial cells . Aetiology ? Epidemiology ? Incidence Persistent publicity and inhalation on the little asbestos fibres, chrysotile as well as most carcinogenic amosite and crocidolite, could cause severe illnesses, together with malignant lung cancer and hMPM.
Tumours have typically been observed in individuals occupationally exposed to asbestos, in their loved ones and in residents who lived close to asbestos factories and mines. The asbestos microfibres are rigid, sharp and resistant to chemical these details and biological degradation; they collect in to the interstitial tissues, accumulate in the lower part of the lungs and finally reach the pleura. The moment, this tumour was uncommon but its incidence grew and it can be nonetheless raising in several nations due to the previous widespread use of asbestos; the prediction is for a additional boost within the up coming decades, primarily in the countries exactly where the usage of asbestos hasn’t nonetheless been entirely banned .
Mortality from hMPM is determined by exposure to this earlystage carcinogen; the latency in between 1st speak to with all the agent and tumour diagnosis is long: between 15 and more than 30 years. Whether or not about 80% of hMPM is usually attributed to asbestos fibre inhalation, exposure to SV40 and radiations are recognized as even further probable carcinogenic cofactors . In Western Europe, 5000 patients each year Quercetin die of hMPM; the highest incidence rates are actually reported in Belgium and Excellent Britain . The incidence is rising up in many produced countries and in Western Europe is anticipated to rise inside the next 15 many years: early projections for the 1995?2029 period foresee a doubling of hMPM each year between 1998 and 2018 in advance of the decline . A 2004 update of mesothelioma trend during the United states of america described that around 2500 individuals per year are diagnosed, 19% of that are ladies, and even more than 70 000 cases are expected to occur in the upcoming 20 years with all the peak this yr .
Throughout the world, the somewhat uncommon incidence is expanding having a peak expected in ten many years.

The identification of CSCs has fostered the identification of molecules with pro-differentiation effects. The pro-apoptotic/pro-differentiative bone morphogenetic protein four sensitizes colon CSCs to 5-fluorouracil and oxaliplatin, and eradicates CSC-derived tumors in animal versions . Likewise, GBM-SCs exposed to BMP4 displayed reduced clonogenic means coupled with an increased expression of neural differentiation markers . Up coming, the two grownup stem cells and CSCs might possibly express high ranges of MDR pumps . This protective technique extrudes from cancer cells a broad range of amphiphilic compounds including taxanes, anthracyclines, and vinca alkaloids. The capability to pump out numerous chemicals is at present exploited for the HOECHST dye efflux assay, a system made use of for CSCs isolation that define them as side population .
Acute myeloid leukemia SP and neuroblastoma SP are recommended reading characterized by a better potential in extruding numerous chemotherapeutic agents compared with all the non-SP. Additionally, long-term exposure of breast cancer cells to doxorubicin resulted while in the acquisition of stem-like and chemoresistant properties , as documented from the overexpression of the two self-renewal-related and MDR-related genes. While to start with and 2nd generation ABC inhibitors failed to demonstrate a clinical advantage, much more potent third generation antagonists have been synthesized and therefore are undergoing clinical advancement . Then again, it’s been demonstrated the SP won’t always enrich for stem-like cells, no less than when evaluating GBM cell lines and principal cells , confirming preceding findings indicating that chemoresistance of GBM-SCs is independent on the activity of ABC transporters .
Quiescence physiologically defends adult stem cells against unsafe insults and avoids the exhaustion of their replicative possible . Likewise, CSCs in most cases exhibit a slow proliferation kinetics as demonstrated by label-retaining approaches, which exposed that label-retaining cells meet the operative criteria to PHT-427 be defined CSCs and survive chemotherapy, not like the non-label-retaining population . Considering chemotherapy is lively towards quickly dividing cells, it will be conceivable that prolonged exit from cell cycle permits CSCs to survive chemo-radiotherapy. Furthermore, it is recognized that a considerable percentage of patients with apparent organ-confined disorder will working experience distant recurrence years later radical surgical procedure and adjuvant systemic treatment.
As a result, it is conceivable that disseminated cancer cells are able to remain quiescent for years , as a result generating CSCs the perfect candidate for explaining this temporal pattern of recurrence. Hematological malignancies are representing the benchmark for the growth of anticancer agents forcing dormant cancer cells to proceed through the cell cycle.

As much as 10 TP53 mutants could be analysed simultaneously. TP53 Mut Assessor is available for the two Mac and Windows environments and may be downloaded through the TP53 web-site. A complete documentation is accessible with all the application. CONCLUSIONS/PERSPECTIVES The evolution in the UMD TP53 database from a flat file describing 300 mutations in 1992 to an integrative resource centre offered from just one internet site parallels the evolution of our expertise on TP53 and its significance in cancer study. Scientific studies of your loss and/or get of activity of TP53 mutants is surely an active area of investigation in the two standard and clinical analysis and is far from staying entirely understood. Two series of recent reviews have revolutionized the present knowledge on TP53 mutations and their relations to cancer. First, Jackson et al.
showed that mice expressing missense TP53 mutants in breast tumours displayed a better response to treatment in contrast with mice harbouring tumours with a wt allele. Tumours expressing mutant TP53 with and without loss with the wt allele behave differently, as those who retain the wt allele were the significantly less responsive to treatment. Even though this you can check here observation contradicts the prevailing viewpoint that TP53 mutations are associated with lack of response to many medicines utilized in chemotherapy, it supports isolated observations in human breast cancer that describe a much better response to treatment for tumours expressing non-functional TP53. Similarly, medullary breast cancers that show a frequency of TP53 mutations of _95% are related by using a good prognosis . Even more investigations will decide whether these observations is often extended to other cancer forms.
A 2nd series of reports showed that TP53 mutants deficient for development arrest, apoptosis and senescence are nonetheless lively for tumour suppression . These scientific studies advised that other pathways such as metabolic regulation or antioxidant perform need to be explored to achieve a better awareness Artesunate on mutant TP53 and its relation to neoplasia. Every one of these research were carried out in mice, and additional research in people will likely be expected to clarify the correct anti-neoplastic perform of the TP53 gene. A significant challenge from your work of Jackson et al. worries information with the status in the TP53 wt allele in human tumours, that is a complicated situation owing to your heterogeneity from the tumour components. NGS will possibly have the capacity to resolve this dilemma by providing a lot more exact data.
Steady integration of those data are going to be essential to provide an accurate picture of this complicated protein. The cancer gene LSDB era will come to an finish as information are now collected and compiled from huge information coordination centres this kind of since the Cancer Genome Atlas and COSMIC .

In assistance of this, we observed a statistically significant reduce within the ranges on the cellular reductant glutathione on NSC319726 therapy of TOV112D cells at one, three and 24 hours . To find out the significance of these redox changes for the exercise of NSC319726 we handled TOV112D cells from the presence from the reducing agent N acteyl cysteine plus the oxidizing agent diamide. We uncovered that 5mM NAC inhibited the apoptotic action of NSC319726 whereas diamide enhanced it . These information suggest that ROS improvements are critical to the apoptotic mechanism of NSC319726 on p53R175 mutant cells. The reactivation of p53 in mouse tumor designs is proven to be a extremely powerful therapeutic system . A number of little molecules are already claimed to reactivate mutant p53, which include CP 31398, WR 1065, PRIMA one and MIRA 1 . Together with the exception of 1 compound, WR1065, all have been recognized employing classic chemical screens .
Common this content chemical screens favor the usage of matched situation management cell lines derived from your identical parental cell line, engineered this kind of that the case cell line carries the molecular alteration beneath consideration. This will be a fundamental caveat, considering the fact that cancers are known for being heterogeneous in nature. Here we demonstrate our methodology to display for compounds manifesting improved sensitivity within a panel of cell lines carrying p53 mutations independently of their various genetic backgrounds and cell style specificity, which can be a alot more practical model of what on earth is observed during the clinic. Applying this methodology to your NCI60 screen we identified 3 compounds through the thiosemicarbazone relatives. Follow up experiments with two of those compounds corroborated the predicted p53 mutant distinct growth inhibitory properties.
It is probable that this methodology may very well be utilized to determine compounds with increased sensitivity in tumor cell lines carrying mutations in other important oncogene tumor suppressor pathways. It is necessary to note that cell viability assays in Inhibitor 1C along with the apoptosis assays in Inhibitor 2A indicate that there read review is definitely an apoptotic mechanism that is definitely independent of p53 mutational status . What initiates this apoptosis is unclear but may well be linked to either a rise in ROS ranges or ribonucleotide reductase inhibition, two reported mechanisms of action for thiosemicarbazones. Non tumor cell lines which has a WT p53 gene showed reasonably minor to no development inhibition by NSC319726 at these same doses, which would argue towards the inhibition of RR as the explanation.
If increased ROS amounts would be the motive, we speculate that this apoptosis could possibly be resulting from the inability to compensate for these oxidative improvements in a cell lacking a practical p53 transcription issue.

Beads had been washed 4 occasions with ice-cold modified RIPA buffer, supernatant was aspirated, then 60 ?L 2X protein sample buffer was extra per sample. Samples were boiled for 5min, centrifuged at 10000 rpm for 1min, and proteins separated by SDS-PAGE as described over. For TGF-? immunoneutralization research, HT-29 cells had been treated with one?ten ?g/mL anti-TGF-? or Ig handle sera for 30 min before stimulation with TNF-? for 15min. Cells have been then harvested and analyzed for phospho-ERK written content as described while in the following. 2.5. Western Blotting. Monolayers had been stimulated with 10 ng/mL TNF-? or 50 ng/mL EGF and harvested in Mono Qbuffer at unique instances. Following sonication for 30 secs, samples were centrifuged at 12000 rpm for 1min to clear away insoluble material and protein concentrations were determined using a industrial Lowry Assay making use of BSA specifications made inMono Q buffer.
Lysate concentrations have been adjusted to be sure selleck chemical hop over to this website even protein loading, mixed with an equal volume of 2X protein sample buffer , 2 ?M microcystin ), boiled for 2 mins, and separated through electrophoresis . Proteins were transferred for 1.five hrs at 400mA in transfer buffer onto a PVDF membrane . Membranes had been blocked for 1 hr employing 3% skim milk and incubated overnight in key antibody. The antibodies put to use had been as follows: anti-ERK-1 , anti-phospho-ERK 1/2 , anti-phosphotyrosine , anti- EGFR, and anti-HER2 . Secondary staining was carried out by using HRPconjugated goat sera distinct for mouse or rabbit Ig as expected followed by chemiluminescent detection using a business reagent following manufacturer?s guidelines .
Comparisons weremade only among samples isolated and transferred with each other onto exactly the same membrane.Various Somatostatin exposures had been finished to ensure that movie was not overexposed. So that you can verify equal loading of protein, all western blots by using phospho-specific antibodies were stripped and reprobed with antibody towards the nonphosphorylated kinase. two.six. TACE Exercise. HT-29 cells had been incubated in serumfree media overnight, washed as soon as with serum-free media, and stimulated with ten ng/mL TNF-? for 15mins. Cells have been washed 2X with ice-cold PBS and harvested on ice. TACE action was measured utilizing a commercially available fluorimetric TACE assay kit as per manufacturer?s guidelines. Fluorescence was measured each and every 5mins for 3 hrs and plotted as time passes. Data represents fluorescence following one hr incubation with fluorescent substrate that is inside the linear portion for all curves.
two.seven. HER2 siRNA Knockdown. Single-cell suspensions of HT- 29 cells had been prepared by trypsinizing 100mm confluent monolayers. five ? 105 cells had been transfected with 80 pmols siRNA reagent working with Lipofectamine 2000 following manufacturer?s guidelines.

Direct labeling and flow cytometric examination exposed that every one of the 6 thyroid cancer cell lines studied express cell surface EGFRs at high amounts . Ordinary thyroid cells showed considerable heterogeneity with respect to the two autofluorescence and EGFR surface expression . Frequency distributions of stained cells versus autofluorescent controls have been significantly various in all situations . Differential mean fluorescence intensity to the malignant cell lines was, on common, greater than those on the normal thyroid samples . Therapy of cells with EGF for 24 h resulted in downregulation of EGFR through the cell surface, suggesting receptor internalization and, hence, the presence of practical receptors . Western blotting confirmed overexpression of EGFR by thyroid cancer cells, which displayed EGFR levels somewhere around twice that of normal controls . EGFR activation stimulates invasion Invasion was assessed 48 h submit therapy for all cell lines.
The follicular and papillary carcinoma cell lines displayed an invasive phenotype in addition to a robust response to EGF , with 1.3 to 7 fold increases in invasion observed . FTC 238 cells displayed reduced baseline invasiveness but were hugely delicate to EGF, as the addition of Paclitaxel ic50 only 1.0 ng ml EGF elicited a significant enhance in invasion . TPC one cells displayed the highest baseline invasiveness and responded to EGF which has a reduce but important increase in invasion . ARO 82 1 cells displayed five ten invasion irrespective of remedy, and XTC 1 cells were found to be minimally invasive . EGF did not appreciably have an impact on cell proliferation or death under the situations put to use, which concerned reduced initial plating densities and a quick time course.
The two untreated and EGFtreated cells displayed exponential development by using a doubling time of 36 48 h, suggesting that the greater invasiveness apoptosis activation brought on by EGF isn’t linked to elevated cell proliferation in our model. Therapy of follicular and papillary carcinoma cell lines with AG1478 abolished EGFstimulated invasion . Invasion by cells treated with EGF AG1478 was not significantly numerous from that by cells taken care of with AG1478 alone, indicating total inhibition of EGFR tyrosine kinase action at this dose. AG1478 diminished invasion by TPC 1 cells to 9 under manage, suggesting the presence of endogenous EGFR exercise at baseline. AG1478 had no effect on cell growth or survival. Both GM 6001 and Col 3 diminished EGF stimulated invasion, although a lot more potent results had been observed with Col three .
Larger doses of GM 6001 have been cytotoxic and tough to achieve resulting from restricted solubility. The result of Col 3 paralleled that of AG1478, with one particular exception: 10 g ml Col three had a higher anti invasive impact in FTC 133 cells. This cell line was alot more sensitive to your cytotoxic effects of Col 3 than the other individuals, as being a dose of 10 g ml caused a 22 reduction in cell mass at 48 h . Col 3 was not cytotoxic on the other cell lines.