Ibuprofen has been reported to possess anticancer activity. In the present work, four ibuprofen conjugates of resorcinarene-Polyamidoamine PAMAM-dendrimers were synthesized with eight or 16 ibuprofen moieties. The ibuprofen was released from the dendrimers in a dependent manner. The drug-conjugated nanoresorcinarene-dendrimers showed higher cellular uptake than free ibuprofen. In vitro cytotoxicity studies were performed with free ibuprofen and with the synthesized conjugates in U251, PC-3, K-562, HCT-15, MCF-7, SKLU-1, and MDA U251 (human glioblastoma), PC-3 (human prostatic adenocarcinoma), K-562 (human chronic myelogenous leukemia cells), HCT-15 , MCF-7 (human mammary adenocarcinoma), SKLU-1 (human lung adenocarcinoma), and MDA-MB-231 (human mammary adenocarcinoma) cancer cell lines by different cytotoxicity assays. Ibuprofen conjugates of the first and second generations showed significant cytotoxic effects towards the human glioblastoma (U251) and human mammary adenocarcinoma  cell lines. Moreover, the ibuprofen conjugates improved cytotoxicity compared to free ibuprofen. Increased therapeutic efficacy was observed with specific ibuprofen conjugates of the second generation using low doses.  The use of this therapeutic agent is restricted due to its side effects such as stomach ulceration, bleeding, and perforation ,2. The tIbuprofen has been used as an anticancer drug by itself or in conjunction with drug carriers such as hydrogels , liposom™ X-100 solution , phosphate-buffered saline pH 7.4 , and ethanol (Sigma-Aldrich) were used without additional drying. Tetrahydrofuran THF was dried with Na. N,N-Dimethylformamide , and dimethylsulfoxide (DMSO) . 2-(4-Isobutylphenyl) propionic acid (ibuprofen), , Resorcinol , hydrocinnamaldehyde , dodecyl aldehyde , hydrochloric acid (36.5–38% Aldrich), ethylendiamine , methyl bromoacetate , and methyl acrylate  were used without additional purification. TritonResorcinarene macrocycles and PAMAM-dendritic arms were synthesized and characterized as reported by Cortez-Maya et al. .Resorcinarene-dendrimers (0.0525 mmol) were dissolved in methanol (40 mL) and heated at 80 °C. After 20 min, ibuprofen (0.63 mmol) was added in methanol. The mixture was stirred and heated at 120 °C for 24 h. The solvent was evaporated and the resulting solid was dissolved in methanol and precipitated by ethyl acetate EtOAc.1H and 13C nuclear magnetic resonance NM spectra were recorded on a Varian Unity-300 MHz with tetramethylsilane (TMS) as an internal reference. Infrared (IR) spectra were measured on a Bruker FTIR tensor 27 Nicolet FT-SSX spectrophotometer . Elemental analysis was determined by Galbraith Laboratories, INC Knoxville. FAB+ mass spectra were taken on a JEOL JMS AX505 HA instrument . Electrospray mass spectra were taken on a Bruker Daltonic, Esquire 6000 . MALDI-TOF mass spectra were taken on a Bruker Omni FLEX  using 9-nitroanthracene (9NA) as a matrix. The ultraviolet-visible UV-vis absorption spectra were obtained at room temperature with a Shimadzu 2401 PC spectrophotometer . 50 values were estimated by fitting the inhibition data to a dose-dependent curve using a logistic derivative equation. U251 (human glioblastoma), PC-3 (human prostatic adenocarcinoma), K-562 (human chronic myelogenous leukemia cells), HCT-15 , MCF-7 (human mammary adenocarcinoma), SKLU-1 (human lung adenocarcinoma), and MDA-MB-231 (human mammary adenocarcinoma) cell lines were supplied by the National Cancer Institute . Human gingival fibroblasts (HGF) were supplied by the National Cancer Institute . Each treatment was performed in heptaplicate wells. The ICThe hydrolysis of conjugates was studied at pH 7.4 ). All experiments were performed in triplicate along with controls. Four milligrams of each conjugate were added into 4 mL of preheated buffer solutions. All of the release solutions containing 1 mg/mL of the conjugate were stirred continuously and maintained at 37 °C. At different time intervals, samples were withdrawn and the reaction was stopped with a methanol solution of sodium salicylate (1.25 mM) and analyzed by UV and IR spectroscopies. A Synergy hybrid technology HT Microplate Reader  quantitatively determined the cellular uptake of ibuprofen-conjugated dendrimers. PC-3 cancer cells were seeded in a 96-well black plate at 10,000 cells/well and cultured. After 15 days, the cancer cells came to 65–80% confluence. The culture medium was replaced by Transport Buffer (pH 7.4). The PC-3 cancer cells were incubated with either ibuprofen conjugates or free ibuprofen at a concentration of 10 μM/mL for 6 h at 35 °C. After incubation, the suspension was removed and cells were washed four times with 50 μL cold phosphate-buffered saline solution (PBS (pH 7.4)). After that, 100 μL of 2% Triton X-100 in PBS were added to the wells to lyse the cells. The fluorescence intensity of each sample well was measured by a microplate reader calibrated with standard solutions of ibuprofen (0.1–50 μM/mL) in similar conditions. The excitation and emission wavelengths were 220 and 410 nm, respectively, for ibuprofen. Experiments were performed in triplicate. 50 for each tested drug were calculated from at least three different experiments. For the assay with Human gingival fibroblast (HGF) cells, the tested compounds were dissolved in fresh culture medium with 2% DMSO to afford different concentrations .Cytotoxicity assays were determined using the protein-binding dye sulforhodamine B (SRB) in microculture to measure cell growth, as described in the protocols established by the National Cancer Institute NCI  [Recently, we showed that resorcinarene-dendrimers can be obtained with a crown conformation of first and second generations, with terminal amine groups that can be functionalized with the acid group of the ibuprofen to obtain the conjugates of first and second generations with eight and 16 ibuprofen moieties .1 was characterized by 1H NMR spectroscopy  spectroscopy. Detection of the hydrolyzing solution by UV spectrophotometry showed that only the ester bond between the drug moiety and the dendritic branches is hydrolyzed during the reaction time, and it was observed that the four ibuprofen-conjugated dendrimers 1–4 showed a relatively slow release in the first days followed by a sustained release in the second week. Ibuprofen was released from the corresponding first-generation 1, 2 dendrimers at 68 and 65%, respectively, and the second-generation 3, 4 dendrimers at 76 and 80% , at 37 °C and pH 7.4). The dendrimers were dispersed in the buffer solution and hydrolysis was performed. The amount of free ibuprofen in the buffer solution was determined by UV (at 220 nm) and IR . Dendrimer conjugates at concentrations of 10 μM were cytotoxic to PC-3 cells. ion rate b. The PConjugate d. It couRecent reports showed that free ibuprofen is not toxic in low doses and has antitumoral effects ,6,7,8. T1–4 was evaluated. All compounds were chosen for evaluation of their biological activity against cancer. We screened in vitro against seven human cancer cell lines: U251 (human glioblastoma), PC-3 (human prostatic adenocarcinoma), K-562 (human chronic myelogenous leukemia cells), HCT-15 , MCF-7 (human mammary adenocarcinoma), SKLU-1 (human lung adenocarcinoma), and MDA-MB-231 (human mammary adenocarcinoma). As a control, we also tested against the human gingival fibroblast (HGF) cell line. The initially obtained cytotoxic screening data (1 showed high inhibition (%) of five cell lines, and the conjugate 2 showed high activity against three cell lines: PC-3, MCF-7-7, and MDA-MB-231. For the dendrimer of second generation 3, the activity was 100% in five cell lines. The dendrimer 4 showed high specificity against the K-562 cell line. The antiproliferative results obtained with the conjugates of ibuprofen 1–4 were compared to ibuprofen on its own (free) and to cisplatin, a reference anticancer drug. The cytotoxic activity of the synthesized resorcinarene-dendrimer-conjugates of ibuprofen ing data  showed t1–4 ibuprofen conjugates. A marked dose-dependent inhibition of cell growth was consistently observed in all five cell lines. The detailed profile of the inhibition of these cell lines by our compounds is shown in 1–4 were: U251 (human glioblastoma), PC-3 (human prostatic adenocarcinoma), K-562 (human chronic myelogenous leukemia cells), MFC-7 (human mammary adenocarcinoma), and MDA-MB-231 (human mammary adenocarcinoma). The dose-response behavior of the conjugates 1 and 3 with undecyl chains in the lower part of the resorcinarene showed that the activity depended on the amount of ibuprofen molecules in the dendrimer and the length of the dendritic arm. This also was observed with dendrimers 2 and 4, which have ethylphenyl groups in the lower part of the resorcinarene. The second generation compound 4 was more active against all cell lines, even at low doses, than the first generation dendrimer 2. The antiproliferative experiments were carried out in order to gain some insight into the anticancer properties that are displayed in vitro. The cell lines were exposed for five days to different concentrations (from 0.1 to 20 μM) of the 1–4 under the same conditions was tested against the human gingival fibroblast (HGF) cell line for five days (4 showed the lowest cytotoxic activity after five days. The cytotoxic activity of the conjugates ive days . Compoun50) values determined for all cell lines  ranged from 0.1 to 5 μM with the conjugates of ibuprofen 1–4. The results are shown in 1 had high activity against human glioblastoma (U251), human mammary adenocarcinoma (MCF-7), human lung adenocarcinoma (SKLU-1), and human mammary adenocarcinoma (MDA-MB-231). The conjugate 2 showed high selectivity against the human chronic myelogenous leukemia cells (K-562) and human mammary adenocarcinoma (MDA-MB-231) cell lines. The conjugate of second generation 3 was very active against all the cell lines studied. The conjugate 4 showed good activity against the five cell lines U251, K-562, MFC-7, SKLU-1, and MDA-MB-231. The inhibitory concentration (IC4, which was found to be the best inhibitor of this series against U251 and K-562, MFC, and MDA cell lines with an IC50 of 3.3 ± 0.02, 3.0 ± 0.5, 3.0 ± 0.3, and 3.1 ± 0.5, respectively. Moreover, these compounds were tested against HGF normal fibroblasts in order to determine their selectivity for cancer cells compared to normal cells. Compound 4 showed higher selectivity for the cancer cells than for HGF fibroblasts. In particular, U251 human glioblastoma, K-562 human chronic myelogenous leukemia cells, MCF-7 human mammary adenocarcinoma, and MDA-MB-231 human mammary adenocarcinoma cancer cell lines appeared to be more sensitive to growth inhibition by the conjugates of ibuprofen than the PC-3, HCT-15, and SKLU-1 cancer cell lines. The most interesting result was obtained with compound 1–4 were synthesized in this work: compound 1 had the corresponding dodecyl group in the lower part of the macrocycle and eight ibuprofen moieties in the structure. Compound 3 also had the corresponding dodecyl group in the lower part of the macrocycle and 16 ibuprofen moieties in the structure. With 16 moieties, compound 3 was more active than compound 1. Compounds 2 and 4 had ethylphenyl groups in the resorcinarene macrocycle and had eight or 16 ibuprofen moieties, respectively. Compound 4 showed better results inhibiting cancer cell proliferation than compound 2. The results of these experiments clearly indicate that 3 and 4, with 16 ibuprofen moieties, inhibit the proliferation of all cancer cell lines tested. The ranked order of potency was 4 > 3 > 1 > 2 at the concentrations assayed.Four resorcinarene-dendrimer-conjugates of ibuprofen In the present work, it was observed that the yields of synthesis to obtain the conjugates of ibuprofen with resorcinarene-dendrimers with aliphatic and aromatic substituents in the lower part of the macrocycle are generally good. The hydrolysis of drug-conjugate dendrimers depends on the length of the dendritic branches and the chemical structure of the dendrimer. These affect the release rate of ibuprofen. The cellular internalization of resorcinarene-PAMAM-ibuprofen dendrimers could occur via clathrin-mediated endocytosis. Biological activity tests showed that the synthesized compounds have high potential activity against cancer. The inhibition of cancer cell proliferation was enhanced in the presence of eight and 16 ibuprofen-moiety-substituents in the dendritic branches compared to ibuprofen alone. The conjugates were slightly cytotoxic to human gingival fibroblast cells and showed improved efficacy over the free drug.


Although the nuclear factor-erythroid 2-related factor 2 (NRF2) pathway is one of the most frequently dysregulated in cancer, it is not clear whether mutational status is a good predictor of NRF2 activity. Here we utilise four members of the aldo-keto reductase (AKR) superfamily as biomarkers to address this question.Twenty-three cell lines of diverse origin and NRF2-pathway mutational status were used to determine the relationship between AKR expression and NRF2 activity. AKR expression was evaluated in lung cancer biopsies and Cancer Genome Atlas (TCGA) and Oncomine data sets.NRF2 pathway. In non-mutant cell lines, co-ordinate induction of AKRs was consistently observed following activation of NRF2. Immunohistochemical analysis of lung tumour biopsies and interrogation of TCGA data revealed that AKRs are enriched in both squamous cell carcinomas (SCCs) and adenocarcinomas that contain somatic alterations in the NRF2 pathway but, in the case of SCC, AKRs were also enriched in most other tumours.AKRs were expressed at a high basal level in cell lines carrying mutations in the An AKR biomarker panel can be used to determine NRF2 status in tumours. Hyperactivation of the NRF2 pathway is far more prevalent in lung SCC than previously predicted by genomic analyses. NRF2 is a cap‘n'collar (CNC) basic-region leucine zipper (bZIP) transcription factor that regulates a diverse battery of cytoprotective genes that collectively allow cells to survive transient periods of exposure to electrophilic, oxidative and inflammatory stress  predominate, while the aldo-keto reductases (AKRs) are more conspicuous when human cells are examined. AKR1B10, AKR1C1, AKR1C2 and AKR1C3 constitute some of the most inducible targets of NRF2 in human systems, both normal and tumour-derived  in both basal and induced states. We show that AKR expression is related to the status of the NRF2/KEAP1 pathway and can be used as a readout for the activation or inhibition of NRF2. Through immunohistochemical analysis of lung tumour biopsies and interrogation of data from The Cancer Genome Atlas (TCGA), we have found that AKRs are enriched in both SCC and AC that contain somatic alterations in the NRF2 pathway but, in the case of SCC, AKR enrichment also occurs in cells not carrying NRF2 or KEAP1 mutation. These data indicate that NRF2 is frequently constitutively activated by alternative mechanisms in this cancer type and, therefore, that genetic analyses alone cannot determine the contribution of NRF2 to the tumour phenotype.In order to examine whether the ability of NRF2 to drive NRF2 or KEAP1, as confirmed by the Wellcome Trust Sanger Institute COSMIC database, was assembled. The origin, authentication and culture conditions of these cell lines are described in detail in KEAP1 , while neither H1395 nor H1993 contain the reported heterozygous mutation . Our sequencing of these cell lines agreed with the Sanger database entries in each case -1, 2, 4a, 6, 8a, 9, 10, 10a-octahydro-8a-ethynyl-1,1,4a-trimethyl-2,6-dioxophenanthrene-3,7-dicarbonitrile (TBE-31) was synthesised as described previously  and non-targeting (D-001810-10-05) siRNA SMARTpools, each containing four siRNAs , at a final concentration of 10 nmol l−1, in complex with Lipofectamine RNAiMAX . Cells were lysed for analysis at the timepoints indicated. For fractionation of nuclear and cytoplasmic compartments, cells were processed using the NE-PER Nuclear and Cytoplasmic Extraction kit (Thermo Fisher Scientific). ELISAs were carried out by the In-Cell colorimetric method (Thermo Fisher Scientific) and statistical significance evaluated by unpaired t-test: *P⩽0.05, **P⩽0.01, ***P⩽0.001.Sulforaphane (SFN) was purchased from LKT laboratories . (±)-, mouse monoclonal anti-GAPDH (ab8245), mouse monoclonal anti-TBP (ab51841) and rabbit monoclonal anti-LDH (ab52488) were purchased from Abcam . Mouse monoclonal anti-AKR1C3 was a kind gift from Professor Trevor Penning, University of Pennsylvania, PA, USA. SDS–PAGE and western blotting were carried out as described previously  according to the manufacturer's instructions. All real-time PCR primer and probe sets were purchased from Applied Biosystems ; AKR1B10 (Hs00252524_m1), AKR1C1/2 (Hs00413886_m1), AKR1C3 (Hs00366267_m1), NRF2 (Hs00975960_m1), Actin B internal standard (4352935E). Assays were performed in triplicate and fold changes calculated using the 2(−δδ C(T)) method. Fold changes for inducer-treated cells were calculated relative to vehicle-treated cells and the heatmap generated using ggplot2 in R. Statistical significance was evaluated using an unpaired https://tcga-data.nci.nih.gov/tcga/). For AC, analyses were performed on the ‘TCGA, Nature 2014' data set. For SCC, analyses were performed on the ‘TCGA Provisional' data set . Normalised level 3 RNA-seq gene expression values, as determined using the RNA-Seq by Expectation Maximisation (RSEM) algorithm, were retrieved using the TCGA-Assembler package in R and RStudio . For the comparison of wild type with mutant non-small cell lung cancer (NSCLC), we analysed 230 cases of AC  and 178 cases of SCC . For the comparison of tumour to matched normal tissue we analysed data for 45 tumour/normal  paired AC and 16 tumour/normal  paired SCC samples. All data processing and statistical analyses were carried out in R using packages cgdsr, plyr and ggplot2. Association was evaluated by unpaired and paired Wilcoxon ranked test for wild-type vs mutant values  and matched normal vs tumour values , respectively.All gene expression and mutation data for AC and SCC were derived from publicly available TCGA datasets  and HO1-u-1 , while AKR1C3 was expressed at low, but detectable, levels in H838 (homozygous for KEAP1 mutation), 5637 and MDA-MB-231 (KEAP1 and NRF2 wild type) cells  cells . It shoue) cells .bis-enone inducer H dehydrogenase, quinone 1 (NQO1), the protein most commonly used as a proxy measure of NRF2 activity, showed that it was constitutively expressed in almost all of the cell lines, being undetectable in only AD293 and MDA-MB-231. NQO1 was inducible in many of the lines homozygous for wild-type KEAP1 and NRF2, but the fold changes were less than those of the AKRs. NQO1 was not inducible in the mutant cell lines.In many of the cell lines homozygous for wild-type  protein . In someThe A549 and H838 cell lines, which lack functional KEAP1 and hence exhibit elevated NRF2 activity , with a KEAP1  or AC (n=7). Positive immunohistochemical staining was detected for at least one AKR in all SCC samples and for two or three AKRs in seven of eight samples (scoring provided in P=0.0002) by two-tailed Fisher's exact test.Fifteen FFPE samples of NSCLC were classified by conventional histological criteria as either SCC  that had low expression of the transcript, approximately equal to the wild-type median level. Case IDs for the 10 samples with the lowest level of expression of each of the four genes under study were compared , strong correlations were observed between AKRs, and of AKRs with other NRF2-regulated genes, such as GPX2, GSR, PGD, G6PD, GCLM, GCLC, ME1, SRXN1 and NQO1  as follows; AKR1B10: 0.002, AKR1C1: 0.002, AKR1C2: 0.004, AKR1C3: 0.002. In SCC, all four AKRs were highly significantly enriched in tumour tissue, compared with normal, irrespective of mutational status, although mutant cases generally possessed a higher relative level of expression. Median fold enrichments in SCC were as follows: AKR1B10: 529-fold, AKR1C1: 16-fold, AKR1C2: 50-fold, AKR1C3: 7-fold. This pattern of enrichment in AC and SCC was also seen with the other NRF2 target genes, GCLC, GCLM and NQO1, although HMOX1 was downregulated in both tumour types . We found a highly significant association of AKR1B10 overexpression with SCC of the lung in multiple analyses; examples from Lee et al . We therefore disregarded this factor in our analyses to maximise specificity for the NRF2 pathway.The NRF2 pathway may become activated in tumours through a large number of mechanisms. Core components of the pathway itself are some of the most frequently mutated genes in cancer, with mutations in The implication of these (and other) observations is that constitutive NRF2 activity is far more common in tumours than has been predicted by the frequency of genomic or epigenomic alteration of the pathway, and that these analyses cannot therefore definitively predict its phenotypic status. In order to effectively interrogate CUL3/KEAP1/NRF2 pathway status, a robust downstream signature of activity is required. We had previously identified AKRs 1B10, 1C1, 1C2 and 1C3 as some of the most inducible targets of the human NRF2 pathway in the spontaneously immortalised keratinocyte cell line, HaCaT  of the remaining mutant lines exhibited high levels of one these enzymes individually. High levels of AKR coincided with high levels of NRF2. With very few exceptions , AKRs were expressed at relatively low constitutive levels in cell lines with non-mutated KEAP1/NRF2. Importantly, the true (relative) level of NRF2 activity in each of these cell lines is unknown. It has been shown, however, that certain KEAP1 mutations have little or no influence on the capacity of KEAP1 to repress NRF2  basal levels of AKR. In the case of H838, it has previously been shown that these cells exhibit lower levels of enzymatic activity of NQO1, the most commonly used proxy for NRF2, than do A549 and H460  of seven lines carrying mutant ta et al  reportedon et al . InteresG12D/Trp53R172H mouse model of pancreatic cancer, with an accompanying decrease in Ras signalling  As all NRF2 target genes will be subject to multifactorial regulation, we suggest that the expression of AKRs should be considered a signature of NRF2 activity. This signature, while generally segregating with NRF2/KEAP1 genotype in cultured cells , did not appear to do so in lung SCC, as high levels of AKR were detected in tumours without these mutations. This finding suggests that additional mechanisms of pathway regulation, potentially those with roots in the environmental or stromal context of the tumour, may often be key determinants of its status in vivo. In addition, our data support the contention that, while the transient activation of this pathway may be chemopreventive in normal cells, its chronic activation in cancer cells confers a selective advantage through enhanced proliferation and chemoresistance.Upon challenge with chemical inducers of NRF2, we observed an increase in mRNA for KEAP1 occur primarily in AC and mutations in NRF2 occur primarily in SCC (NRF2 mutations are associated with squamous carcinomas irrespective of the tissue of origin. In one study of 1145 carcinoma samples from 13 tissue types, of which 167 were squamous, 21 of the 22 detected mutations in NRF2 were in SCC (CUL3/KEAP1/NRF2 pathway was detected in 34% of 178 cases (AKR1B10 mRNA transcript levels in SCC and AC of the lung (NRF2, increased AKR levels segregate to SCC of tissues other than lung. Future work could also incorporate other AKRs that are known to be regulated by NRF2 but have not been included in this study, such as AKR7A2 (Non-small cell lung cancer is the tumour type in which KEAP1/NRF2 dysfunction has most frequently been reported. Within NSCLC, mutations in 78 cases . PertineAKR1B10, AKR1C1, AKR1C2 and AKR1C3 are transcriptional targets strongly indicative of NRF2 status in human tumours, and that NRF2 is the upstream effector of AKR overexpression in cancer, particularly in tumours of squamous origin. Although a large number of studies have identified genetic, epigenetic, signalling and other changes that can lead to NRF2 hyperactivation in both AC and SCC of the lung, our data indicate that this is of more widespread downstream consequence in SCC and, moreover, this hyperactivation frequently occurs in the absence of any somatic mutation of the pathway. If, as is widely thought, activation of NRF2 supports tumour growth and proliferation, targeted inhibition of the NRF2 pathway may constitute a potential therapeutic avenue in most cases of this cancer type. In conclusion, therefore, the utilisation of AKRs as biomarkers of NRF2 status will ultimately help determine the significance of this pathway in cancer genesis and progression, and of its potential roles in disease management and the design of therapeutic strategies.The present work demonstrates that


In this study, we examined 12 HSCT recipients with GVHD-caused DED and 10 HSCT recipients without GVHD-associated DED and evaluated the clinical parameters in the 2 groups. Analysis of the central cornea and limbal epithelia using IVCM was conducted to investigate the density of the corneal sub-basal nerves, DCs and GICs as well as the tortuosity and branching of the sub-basal nerves. As suggested by our data, the clinical variables in the GVHD group were significantly different from those in the non-GVHD group. Additionally, GVHD-triggered DED conceivably increased the density of DCs and GICs in the central cornea and the density of DCs in limbal epithelia and altered the morphology of the sub-basal nerves. These phenomena are presumably correlated with the degree of inflammation. Thus, our findings may be translated into non-invasive diagnostic methods that indicate the severity of inflammation on the ocular surface in HSCT recipients.Dry eye disease (DED) is often elicited by graft-versus-host disease (GVHD), an extensive complication of hematopoietic stem cell transplantation (HSCT). To unravel the mechanism of this type of DED,  However, medical settings are still required to combat disabling complications resulting from HSCT. One of the most notorious disorders is graft-versus-host disease (GVHD), which can affect multiple organs. GVHD is one of the leading causes of morbidity after HSCT and occurs in 10–90% of HSCT recipients4. The eye is one of the most GVHD-vulnerable organs, and 40–60% of patients who undergo HSCT develop ocular GVHD3. In many cases, patients with ocular GVHD suffer from ocular disorders, visual deterioration, lacrimal gland dysfunction, meibomian gland dysfunction, corneal epitheliopathy, conjunctival scarring, and hyperemia. These conditions induced by ocular GVHD have a large detrimental impact on patients’ quality of life6.Hematopoietic stem cell transplantation (HSCT) is widely used for the treatment of hematological malignancies and several benign hematopoietic diseases. Technology has progressed considerably during the last decades, and consequently, the success rate of HSCT itself has substantially improved8, the exact pathogenesis of ocular GVHD remains to be elucidated. Previous studies suggest that donor-derived immunocompetent cells attack host tissues and presumably play a contributory role in the development of GVHD10. Because there are limited data evaluating the ocular surface in GVHD patients, an investigation into GVHD-elicited changes of the ocular surface at the cellular level could provide some insights into the developmental process that leads to ocular GVHD.Although several effective medications have emergedIn vivo confocal microscopy (IVCM) provides high-resolution images in a non-invasive manner and enables an examination of the ocular surface at the cellular level. In many previous studies, ICVM has been used to assess cellular and structural changes of the ocular surface in patients with various diseases, such as herpes simplex virus (HSV) keratitis, corneal graft rejection, rosacea and dry eye disease (DED), especially chronic GVHD-related dry eye disease14. GVHD-induced changes in the state and/or behavior of corneal sub-basal nerves, corneal epithelial dendritic cells (DCs), and conjunctival epithelial immune cells have been reported14. In this study, the status of the ocular surface in HSCT recipients with GVHD-induced DED was compared with those without DED. Considering that GVHD patients undergo total body irradiation (TBI), usually receive immunosuppressive therapies and have underlying hematopoietic diseases, alterations in the condition of the ocular surface may be induced by these factors15. This clinical study comprehensively focused on these matters. Consequently, the results may potentially elucidate the link between GVHD-triggered DED and the deterioration in the state of the ocular surface in HSCT recipients.Twenty-two eyes from 22 HSCT recipients  with GVHD-related DED (n = 12) and without GVHD-related DED (n = 10) were examined. The patients’ characteristics in both groups are shown in Table Primary hematological diseases in the GVHD group were acute myeloid leukemia , myelodysplastic syndrome , acute lymphocytic leukemia ALL, n = 2), chronic myeloid leukemia  and peripheral T-cell lymphoma , and those in the non-GVHD group were AML (n = 3), Hodgkin lymphoma , CML (n = 1), ALL (n = 2), chronic myelomonocytic leukemia , aplastic anemia  and non-Hodgkin lymphoma . There was no significant difference in age range (p = 0.473), gender diversity (p = 1.000), time-period after HSCT (p = 0.660), donor type (p = 1.000), systemic immunosuppressive treatment  or ratio of TBI (p = 0.096) between the GVHD and non-GVHD groups. The topical medications used by the patients are shown in Table , chronicClinical variables and IVCM parameters are depicted in Table 2)20. Kheirkhah et al.13 reported that the density of sub-basal nerves in patients with GVHD-caused DED was 16.3 ± 6.1 mm/mm2, a value similar to ours and that supports the accuracy of our data. Our literature review revealed that the density of the sub-basal corneal nerve is thought to be positively correlated with the corneal sensitivity22. Indeed, our earlier work also demonstrated (1) a decrease in corneal sensitivity in HSCT recipients with and without ocular GVHD-induced DED and (2) that there was no significant difference in corneal sensitivity between the two groups23. Our new data indicating the decreased density of sub-basal corneal nerves in both GVHD and non-GVHD groups is therefore consistent with that presented in our previous study. Based on these observations, the conditioning regimen that HSCT recipients undergo may well be one of the factors that decreases the sub-basal nerve density. Other research groups have reported that the nerve density in patients with and without Sjögren’s syndrome (SS) is not only decreased due to DED25 but is also affected by other autoimmune diseases such as rheumatic arthritis, which is not associated with SS26. However, we found no significant difference in sub-basal nerve density between the GVHD and non-GVHD groups.Corneal innervation is one of the most important aspects indicating the state of the ocular surface. As evidenced by previous studies using IVCM, various diseases can alter the density, tortuosity and branching of corneal nerves. As indicated by our study, the density of sub-basal nerves in both GVHD and non-GVHD groups was lower than that in normal eyes  compared with their counterparts without these conditions. In contrast, another study suggested that the degree of nerve branching in DED patients was lower in comparison to that in normal subjects22. Lastly, several groups reported no significant difference in nerve branching between patients with DED and normal control subjects25.In addition to the tortuosity, we investigated the branching of sub-basal nerves. Interestingly, our observations suggested that the sub-basal nerves in the GVHD group were branched at a greater level compared with those in the non-GVHD group, while Steger and colleagues31. Thus, the difference in the stage and severity of DED and the degree of localized inflammation caused by DED presumably account for the inconsistent findings of several research groups about changes in the sub-basal nerves. There is sparse information about the exact mechanisms underlying the morphological changes in sub-basal nerves induced by ocular surface diseases. Furthermore, it is unknown whether the alterations are linked with clinical signs and symptoms of ocular surface disorders. However, our study has provided a clue to solve these riddles. The tortuosity of sub-basal nerves and the level of nerve branching appeared to be increased in the GVHD group. It is therefore conceivable that GVHD-related DED induces morphological alterations in the sub-basal nerves, which promotes severe inflammation on the ocular surface in HSCT recipients. The degree of the morphological changes is presumably an indication of the extent of inflammation that has occurred on the ocular surface.The increased tortuosity and branching of sub-basal corneal nerves are thought to result from nerve regeneration caused by the simultaneous action of damaging phenomena and nerve growth factors (NGF) and/or cytokines secreted on the ocular surface during the inflammatory process38. Therefore, we evaluated the behaviour of DCs in the central cornea and limbal epithelia in HSCT recipients with or without GVHD-related DED using IVCM. The literature suggests that IVCM has several advantages that enable uncomplicated detection and quantification of DCs in the central cornea and limbal epithelia39. However, IVCM allows us to detect other kinds of cells in addition to DCs and can only provide information on the morphology and location of cells. Therefore, identifying DCs only based on morphology can be scientifically inadequate. Macrophages, for instance, may also have a dendritic shape41. However, because they are reported to be found exclusively in the corneal stroma41, we therefore identified cells with dendrites in epithelia as DCs.DCs, which are also referred to as Langerhans cells (LGs) in the epidermis and mucosal membranes of the ocular surface, act as main APCs on the ocular surface and play a vital role in maintaining immune homeostasis in the eyes. Additionally, they are known as key players in the initiation of immune responses2) than those in the non-GVHD group (37.57 ± 21.72 cells/mm2). One of the novelties of our clinical study was that we compared the density of DCs in HSCT recipients with and without GVHD. Consistent with our finding, Kheirkhah et al. previously reported a similar density of DCs in the central corneas of GVHD patients (146.71 ± 93.36 cells/mm2)14, supporting the accuracy and dependability of our clinical examination. Our results also indicated that the density of DCs in the limbal epithelia in HSCT recipients with GVHD was considerably greater than that in those without GVHD (116.50 ± 52.61 vs. 55.57 ± 34.24 cells/mm2). These outcomes are presumably indicative of the higher degree of immune activation and inflammation on the ocular surface in GVHD patients and suggest that the changes in DCs in the central cornea and limbus in HSCT recipients with DED are induced by ocular GVHD instead of the conditioning regimen prior to HSCT.In this clinical investigation, the central corneas in the GVHD group had a higher density of DCs  and DED patients who did not receive HSCT and were therefore without chronic GVHD (n = 21). Then, they compared IVCM variables in the two groups. As the clinical severity of DED in the GVHD group was significantly greater than that in the control group, they made adjustments to the clinical severity of DED in order to prevent the difference in the severity of DED between the two groups from affecting IVCM variables. After the adjustments, there was no significant difference in IVCM variables between DED patients with GVHD and those who did not undergo HSCT and were therefore free of GVHD14.According to recent studies, DED is presumed to be one of the factors that can increase the density of DCs in corneal epitheliaBecause of the close link between the propagation of GVHD and the progression of DED, a painstaking investigation will be required to distinguish the separate effects of these two events on the changes observed using IVCM. However, as indicated by this clinical investigation, the density of corneal epithelial DCs in HSCT recipients with GVHD was greater than those without GVHD, and the IVCM parameters in the cornea were associated with various clinical variables. Therefore, the density of corneal DCs could be a reliable indicator of the severity of ocular surface damage and inflammation in GVHD patients. Additional studies are needed to corroborate this notion.11 and DED42. The density of GICs in the central cornea in the GVHD group was significantly higher than that in the non-GVHD group. The increased infiltration of GICs may be indicative of a more active immune response. However, it is still unknown why GICs infiltrate into the ocular surface and how they function in that capacity. It is challenging to unravel the phenotype of these types of cells through morphological investigation using IVCM because they do not have characteristic shapes. Continuing endeavours are needed to uncover the phenotype and function of GICs and their role in the pathophysiology of GVHD-related DED.We also focused on GICs in the basal epithelial and sub-basal layers, which have been presumed to play a role in inflammatory ocular diseasesThis clinical study had several limitations. First, the relatively small number of samples in this study did not allow us to comprehensively investigate the difference in IVCM variables between the different severities of GVHD-related DED. We were also unable to subdivide the 2 main groups (GVHD and non-GVHD groups) according to other clinical aspects such as whether HSCT recipients did or did not undergo TBI. Second, although the shapes of DCs presumably reflect the state of activation, maturation and antigen-presenting capability of DCs, we did not explore certain morphological characteristics of DCs, such as their size, the number of dendrites and the dendritic field. More work is required to determine the nature of the correlation between the morphological alterations of DCs and the extent of inflammation induced by GVHD-related DED. In addition, only the inferior limbal epithelia in HSCT recipients with and without GVHD were assessed in this study. Ideally, four quadrants of the limbus should be evaluated in order to comprehensively examine the limbus. However, prolonged examinations are needed to achieve this and would require placing the patients, especially those with GVHD, in very uncomfortable situations. Clinical characteristics such as age range, gender diversity, primary diseases, history of TBI and types of HSCT in the GVHD group were not exactly the same as those in the non-GVHD group. In reality, it is extremely difficult to match the GVHD and non-GVHD groups with respect to the above aspects, since the recruitment of HSCT recipients, especially those without GVHD-related DED, is highly demanding.In summary, the density of DCs in both the central cornea and limbal epithelia in HSCT recipients with GVHD-related DED was significantly higher compared with those without these conditions. In addition, the sub-basal corneal nerves in HSCT recipients with GVHD-related DED appeared to be more vulnerable to morphological changes in comparison with those in HSCT recipients without GVHD-related DED. All in all, these phenomena discovered through IVCM are presumed to be associated with the status of ocular GVHD-elicited inflammation in the eyes. Our clinical study has the great potential to promote the development of diagnostic technologies to monitor the state of inflammation on the ocular surface in HSCT recipients.This study was approved by the Institutional Review Board and Ethics Committee of Keio Hospital (IRB No.: 20130013) and complied with the tenets of the Declaration of Helsinki. The individual participants were notified of all possible consequences of this study, and written informed consent was obtained from each of the subjects.The GVHD group had 12 HSCT recipients with GVHD-related DED who survived for more than 100 days after the transplantation, and the non-GVHD group had 10 equivalents without GVHD-related DED. The patients were 20 years or older when they were recruited.50. Exclusion criteria were: (1) inability to undergo an IVCM examination due to poor physical and/or mental states, (2) written informed consent was not obtained, (3) other ophthalmic diseases such as cataract, retinal detachment and diabetic retinopathy, (4) ocular surgery performed within 6 months prior to the start of this clinical study, (5) current or previous use of contact lenses, (6) signs and symptoms of DED before HSCT, and (7) presence of fundus hemorrhage or uveitis.The subjects in the GVHD group met the Japanese diagnostic criteria for dry eye. The diagnostic criteria are as follows: (1) disturbance of the tear film (Schirmer test ≤ 5 mm or tear film breakup time ≤ 5 seconds); (2) conjunctivocorneal epithelial damage ; and (3) dry eye symptomatology. Patients were diagnosed with definite dry eye only if they met all 3 diagnostic criteria51 and ICOGs52.In this study, the following data and information were collected from the individual subjects: primary hematological diseases, types of HSCT, TBI history, systemic immunosuppressive therapies, topical medication, post-transplantation period, duration after the onset of GVHD-related DED, OSDI, Schirmer’s test without anesthesia, BUT, CFS, CLGS, MGDs, hyperemia, JDEsin vivo confocal laser scanning microscopy . At the beginning of examination, the proper volume of the topical anesthetic 0.4% oxybuprocaine was administered to the conjunctival sac. A drop of gel , which served as a coupling medium, was placed on the anterior surface of a polymethylmethacrylate (PMMA) cap . The individual subjects were instructed to place their heads in a head holder and continue looking forward  or looking up . Then, the examiner placed an applanate PMMA cap onto the area of interest, and the state of the area covered by the PMMA cap was examined through real-time video images displayed on a computer screen. The target areas and reference layers were determined by adjusting a controller and a zoom gear. Once all required adjustments were made, the examiner took images by pressing a foot pedal. Under the “sequence model”, this machine provides automatic collection and storage of 100 images (400 × 400 μm in size) per sequence scan. While operating this machine, the examiner adjusted the focal plane to ensure that all target layers of each target area were clearly recorded. To avoid overlap, three non-overlapping scans in each area were performed.Central cornea and inferior limbal epithelia in all patients were examined using www.imagescience.org/meijering/software). DCs, which have branching dentritic structures, were identified morphologically, and their cellular images were bright , and the average of the scores given by the two observers was shown as the value of each image.The density of DCs and GICs was defined as the number of cells per mm2 test was used for the comparison of qualitative variables. A correlation between IVCM variables and clinical variables was assessed using Spearman’s rank-order correlation test. All p values were 2-sided and considered statistically significant when less than 0.05.Statistical analysis was performed using SPSS software 22 . Continuous variables were reported as the mean ± standard deviation (SD). Categorical variables were presented as frequency or percentage. The Shapiro-Wilk test and the Levene test were conducted to investigate the distribution of data and homogeneity of variances in the data, respectively. Student’s t-test was used to compare the means of the data with a normal distribution and homogeneity of variances in the GVHD and non-GVHD groups. The Welch separate variance estimation t-test was conducted for the data with a normal distribution but without homogeneity of variances. The Mann-Whitney U test was used for the comparison of data with skewed distribution. The χ


Misidentification or cross-contamination of cell lines can cause serious issues. Human cell lines have been authenticated by short tandem repeat profiling; however, mouse cell lines have not been adequately assessed. In this study, mouse cell lines registered with the JCRB cell bank were examined by simple sequence length polymorphism (SSLP) analysis to identify their strains. Based on comparisons with 7 major inbred strains, our results revealed their strains in 80 of 90 cell lines. However, 12 of the 80 cell lines (15%) were found to differ from registered information. Of them, 4 cell lines originated from the same mouse, which had been generated through mating between two different inbred strains. The genotype of the mouse sample had not been examined after the backcross, leading to strain misidentification in those cell lines. Although 8 other cell lines had been established as sublines of a BALB/c cell line, their SSLP profiles are similar to a Swiss cell line. This affects differences in genotypes between inbred and outbred strains. Because the use of inbred samples and interbreeding between strains are not involved in human materials, our results suggest that the cause and influence of misidentification in mouse cell lines are different from those in human.The online version of this article (doi:10.1007/s11626-016-0104-3) contains supplementary material, which is available to authorized users. Misidentification or cross-contamination of cell lines is a serious problem, leading to erroneous research . C57BL/6, BALB/c, DBA/2, 129, and C3H mice obtained from CLEA Japan , A and CBA from Japan SLC  were used as reference panels. All animal experiments were performed in accordance with protocols approved by the Institutional Animal Care and Use Committees of National Institutes of Biomedical Innovation, Health and Nutrition.A list of cell lines examined in this study is shown in Table 6 cells using AllPrep DNA/RNA Mini Kit  or from a short piece of mouse tail using DNeasy Blood and Tissue Kit (QIAGEN). SSLP analysis was carried out at 7 loci using primer sequences available from the Mouse Microsatellite Data Base of Japan (http://shigen.nig.ac.jp/mouse/mmdbj) listed in Table Genome DNA was isolated from approximately 5 × 10Reference SSLP sizes for 6 loci in the 6 inbred strains Tables  and 3 shOur SSLP profiles of mouse cell lines were compared with the reference data to identify the strains of their origins Table , summariFour cell lines  had been deposited as of C57BL/6 origin. However, SSLP profiles of these cell lines show mixed profiles between C57BL/6 and C3H, indicating that they were derived from an intercrossed individual Table . Our inqEight cell lines had been believed as subclones of the BALB/3T3 clone A31 cell line derived from the BALB/c strain  before they are used. SNP analysis has been developed for authentication of mouse cell lines Figure S2(PDF 120 kb)Table S1(XLSX 10 kb)Table S2(XLSX 18 kb)


Reactive oxygen species (ROS) are common products of mitochondrial oxidative phosphorylation, xenobiotics metabolism and are generated in response to several environmental stress conditions. Some of them play important biochemical roles in cellular signal transduction and gene transcription. On the other hand, ROS are known to be involved in a wide range of human diseases, including cancer. The excessive production of such ROS together with disruption of homeostasis detoxifying mechanisms can mediate a series of cellular oxidative stresses. The oxidative stress of redundant free radicals production can lead to oxidative denaturation of cellular macromolecules including proteins, lipids and DNA. Moreover, oxidative damage is one of the major causes of DNA mutations, replication errors and genomic abnormalities which result in either inhibition or induction of transcription, and end with the disturbance of signal transduction pathways. Among affected signaling pathways are redox-sensitive kinases. The stimulation of these kinases induces several transcription factors through the phosphorylation of their module proteins. The activation of such pathways induces proliferation and cellular transformation. A diet rich in antioxidant compounds has potential health benefits, and there is a growing interest in the role of natural antioxidants in nutrition for prevention and cure of cancer diseases. A controversy has risen regarding the relation between antioxidants and the significant decrease in the risk of cancer incidence. In this review, we will focus on redox-sensitive kinases signaling pathways, highlighting the effects of dietary antioxidant on the prevention, incidence, prognosis or even treatment of human cancers. In addition, we will place emphasis on the chemical classes of pterocarpans as natural anti-oxidants/cancers as well as their underlying mechanisms of action, including their effects on MAPKs and topoisomerase activities. Reactive oxygen species (ROS) are known to be involved in a wide range of human diseases, including cancer. The oxidative damage that ROS cause can lead to DNA mutations which result in (either) (i) transcriptional inhibition or induction; (ii) enhancement of signal transduction pathways; (iii) replication errors or (iv) genomic abnormalities. ROS can be defined as a heterogeneous group of molecules that are, along with endogenous antioxidants, ubiquitously present. They are produced mainly in the aerobic cells. Mature myeloid cells generate ROS during innate immune responses. Low levels of ROS regulate normal cell proliferation as well as cell signaling . TherefoOxidative stress has been shown to be implicated in various clinical complications including malignant transformations . ROS canTwo main protein families are known to regulate the ROS-activated signal transduction pathways, namely: the mitogen activated protein kinase (MAPK) and the redox sensitive kinases. Here, we will shed more light on the MAPK. The three-kinase signaling module system consists of MAPK, MAP2K and MAP3K. MAPKs include the growth factor-regulated extracellular signal-related kinases (ERKs), which are subdivided into ERK1 and 2 isoforms, and the stress-activated MAPKs. The stress-activated MAPKs can be categorized into c-jun NH2-terminal kinases (JNKs), which are subdivided into JNK1, 2 and 3 isoforms, and p38 MAPKs, which are subdivided into α, β, γ, and δ isoforms  4]..4].MAP Kinases (MAPKs) are a family of dual-specificity serine/threonine protein kinases, which mediate the transduction of extracellular signals from the cell membrane to the nucleus. They are also implicated in multiple cellular functions, ranging from cell survival and proliferation, to cell differentiation and programmed cell death . After bThe MAP kinases family includes three members: MKK1/2, MKK3/6, and MKK4/7. ERK and p38 are activated by MKK1/2, and MKK3/6, respectively . HoweverMAP kinase phosphatases (MKPs) are a large family of dual-specificity phosphatases . They caMKPs contain two main domains, namely a kinase-binding domain and a phosphatase domain. In the absence of a specific substrate, MKPs show low phosphatase activity. However, the enzymatic activity appears after binding to a specific MAPK in a process called substrate-induced activation mechanism. These two domains play an important role in the tight regulation of substrate specificity and enzymatic activity of the (MKPs) . SeveralMKPs have high affinity to MAPKs but they have different substrate specificity for the MAPK family members. The MKP family can be subdivided into three groups ,12,16,17Several studies classified MKP-1 as a stress-activated gene ,15, whil2O2 enhances MAPK pathways, which indicates the role of ROS in the MAPK activation chromene) which is the main skeleton of pterocarpans and  or 6a hydroxypterocarpans. Goel and coworkers (2012) classified these two main classes of pterocarpans into three subclasses [Pterocarpans were divided into two main classes .The third class is furanopterocarpans: in this subclass furan ring is fused to pterocarpan molecule comparable to pyranopterocarpans these compounds are rarely seen in nature, ex. neodulin  exhibited antiproliferative activity in dose-dependent manner against human(A2780) ovarian and (H23) lung cancer cells lines after 24 h incubation [Zhoh and coworkers (2009) reported that pterocarpan trifolirhizin  reported that chronic inflammation can lead to cancer and the inflammatory mediators as TNF, interleukins, chemokines, COX-2, 5-LOX, and MMP-9 are involved in many steps of the process of tumorigensis [Herein, this study trifolirhizin also showed dose-dependent suppression of LPS-stimulated TNF-α, IL-6, and COX-2 in mouse macrophages, it could be concluded that trifolirhizin, which belongs to rigensis  which isrigensis .2; 3; 4; 5; 50 values were estimated after 3, 6, 12, 24, 36, 48, 60 and 72 h of incubation. The results revealed that compound 3 is more cytotoxic than compound 5 in the three tested cell lines (CEM), (HL-60) and (HCT-8) with IC50 values  respectively. Compound 3 showed a two-fold increase in activity in (HL-60) and (HCT-8) cell lines in comparison with compound 5. Compound 2 has potent cytotoxicity against all tested cell lines with IC50 values  respectively, and it was considered a promising cytotoxic molecule. It could be concluded that methoxy substitution on C-2 in homocarpin and compound 2 are responsible for stronger cytotoxicity [The anticancer and the mode of action of 9-methoxypterocarpans derivatives were studied tentatively by Militao and coworkers in series of studies ,60,61. Ttoxicity .4; Moreover, the mode of action of medicarpin , explored the ability of 9-methoxypterocarpans derivatives to induce apoptosis as part of their cytotoxicity using human promyleocytic leukemia HL-60) cell line [ cell lin2 was evaluated by the trypan blue dye exclusion test against panel of four leukemic cell lines JURKAT, HL60, K562 and MOLT-4, the IC50 values were 8–18.8 µM these results indicated that compound 2 exhibited its effect in both a dose- and time-dependent manner where significant results were obtained after 48 h and 72 h with IC50 values of 0.3 to 2.5 µM, 0.3 to1.6 µM respectively [In a comparative study the antiproliferative effect of compound ectively .2 on the cell cycle progress and microtubule function in three breast cancer cell lines MCF7, T47d and HS578T [2 at concentration 8 µM in a time-dependent manner where a 24 h incubation period followed by a 24 h recovery period in medium free pterocarpan led to a reversible effect while persistent mitotic inhibition followed by apoptosis was noticed after a 48 h exposure period despite the pterocarpan free medium recovery period. In this study, the mitosis was inhibited during prometaphase, in a crucial step where the separation of duplicated centrosomes was blocked followed by cell cycle arrest, and the persistent prometaphase arrest resulted in apoptosis [In recent in-depth study, Militao et al. (2014) studied the anticancer mechanism of compound d HS578T . The celpoptosis . 8; 9; 10; 7; The same antimitotic assay was employed by other authors in another study to evaluate the antimitotic effect of prenylatedpterocarpans cabenegrinsA-I (compound 723) is the binding site where topoisomerases start their nucleophilic attack and form DNA cleavable complex [11; Bituminaria bituminosa flower (Leguminosae) showed antitumor activity against two human adenocarcinoma cell lines (HT29 and LoVo) proficient and deficient in MMR (mismatch repair), p53 and Bcl-2 [11 revealed that it demonstrates its effect through inhibition of topoisomerase I function but in a manner different from the other common topoisomerase I inhibitors. Usually, topoisomerase inhibitors are divided into catalytic or posions. The posions inhibitors bind reversibly to the enzyme-DNA complex after the step of DNA cleavage and delay the DNA relegation step, which induces cell death. The catalytic inhibitors, on the other hand, interfere with one of other steps of the topoisomerase I cycle. Compound 11 exhibited irreversible biding to DNA-enzyme complex, thus completely inhibiting the relegation step and also binding to the topoisomerase I itself. The molecular modeling study revealed that the preferential binding site of compound 11 on the enzyme come within proximity of tyrosine (Tyr723) active site, and its prenyl substitution on C-8 position come in close contact with two active sites residue, (Arg488) and (His632). These are involved in the catalytic reaction of the enzyme on the DNA strand, inhibiting their function so that it blocks the DNA cleavage step. Another interesting finding from the modeling study is the binding of compound 11 to the enzyme which gave enough space fitting for the DNA substrate into the enzyme cavity, so that compound 11 is considered the first example of a natural compound able to inhibit topoisomerase I reaction in both the cleavage and relegation steps without inhibition of the enzyme binding [As mentioned before, both topoisomerases I and II are responsible for cleavage of DNA. While topoisomerase I is able to cleave one DNA strand, topoisomerase II cleaves both DNA strands. However, their function is essential for almost all DNA metabolism processes, and tyrosine  in one study [Erythrina excelsa (Leguminosae) in another study [+/+) colon cancer cells and its knockout clones (HCT116) (p53−/−), the (U87MG) glioblastoma cells and its resistant subline epidermal growth factor receptor (U87MG. ΔEGFR) and human hepatocellular carcinoma cells (HepG2) and the normal hepatocytes (AML12). Both pterocarpans Sophorapterocarpan  cells demonstrated that both of them induced cell cycle arrest in the Go/G1 phase. Compound 14 induced cell cycle arrest between Go/G1 phase and S phase in a time-dependent manner. It has been proven from the results that the tested pterocarpans induces apoptosis in leukemia (CCRF-CEM) cells via different modes of action. Compound 13 was able to increase the activity of caspases which are responsible for cutting the cellular proteins at specific Aspartate residues to regulate the process of apoptosis, compound 13 activated the initiator caspase 3/7 and effectors caspases 8 and 9 two-fold in concentration range (0.5–2-fold IC50), while compound 14 activated caspases 3/7 more than caspases 8 and 9 at a concentration of 2-fold IC50. In another method, compound 12 induces apoptosis through the breakdown of mitochondrial membrane potential (MMP) the disruption of which is a common event in the process of apoptosis by (17%–92.9%) in concentration range form (1/4-fold IC50 to 2-fold IC50). Similarly, compound 14 displayed dose-dependent disruption of MMP (89% at concentration of 2-fold IC50).Kuete and coworker (2014) provided a mechanistic study for the antiproliferative effect of two pterocarpans Sophora pterocarpan A and 6α-hydroxyphaseollidin which were isolated from the bark of ne study , and forer study . The cytErythrina abyssinica (Leguminosae) against the panel of four breast cancer cell lines including drug resistance types [15; 50 values (5.6 to 11.8 µM), it exhibited 2-fold activity of tamoxifen (the reference drug) against the drug-resistant cell lines, tamoxifen-resistant MCF7 (MCF7/TAMR) and adriamycin-resistant MCF7 (MCF7/ADR) cell lines with IC50 (6.2 ± 0.2 and 5.6 ± 0.7 µM), respectively. It has been proven that the presence of 2,2-dimethypyran substitution in ring D of the pterocarpan molecule is the most important feature of the cytotoxic activity against breast cancer cell lines where the presence of this moiety in erybraedin B  cells which also indicates the ability of induction necrosis. Furthermore, it was found that compound 18 significantly breaks DNA strands while compound 20 showed no activity [Furthermore, Nguyen and coworkers (2009) analyzed the cytotoxic activity of 15 isolated pterocarpans from stem bark of ce types . The rest cancer . The genactivity . AdditioA controversy has arisen regarding the relation between antioxidants and the significant decrease in the risk of cancer incidence. Additionally, potent flavonoid-pterocarpans were proven to be potent and promising moieties for inhibition of cancer incidence. However, excessive screenings of the effects of dietary anti-oxidant/cancer pterocarpans on the prevention, incidence, and prognosis, or even the treatment of human cancers are still needed.


Cell lines are still a tool of choice for many fields of biomedical research, including oncology. Although cancer is a very complex disease, many discoveries have been made using monocultures of established cell lines. Therefore, the proper use of in vitro models is crucial to enhance our understanding of cancer. Therapeutics against renal cell cancer (RCC) are also screened with the use of cell lines. Multiple RCC in vitro cultures are available, allowing in vivo heterogeneity in the laboratory, but at the same time, these can be a source of errors. In this review, we tried to sum up the data on the RCC cell lines used currently. An increasing amount of data on RCC shed new light on the molecular background of the disease; however, it revealed how much still needs to be done. As new types of RCC are being distinguished, novel cell lines and the re-exploration of old ones seems to be indispensable to create effective in vitro tools for drug screening and more.The online version of this article (doi:10.1186/s12943-016-0565-8) contains supplementary material, which is available to authorized users. Cell line-based research had a major impact on the development of cancer treatment allowing an innumerable amount of effective drugs to be introduced into practice . Cancer Renal cell cancer (RCC) research has greatly benefited from cell line studies. Cell biology studies enable an understanding of RCC biology and translational studies –15. To aVLH, cMET and TP53 and a general marker immunohistochemistry profile may serve to define the histotype of RCC cell lines  derived cell lines are used as in vitro RCC models, and it is important to remember that cell lines are in fact genetic models of their parent tumor histology . Differees Table . MoleculMajor improvements in the pathologic classification of RCC have been reported over last 30 years. The first, known only as renal cell carcinoma, was in the 1960s divided into clear cell and granular histotypes. Currently five traditional and well-defined subtypes of RCC are known: conventional clear cell RCC, papillary (types 1 and 2) RCC, chromophobe RCC, carcinoma of the collecting ducts of Bellini, and unclassified RCC and these subtypes represent the majority of RCC cases diagnosed . Clear cMET) and fumarate hydratase (FH) alterations are the hallmark of these cancer syndromes, but are infrequent in sporadic cases . Th. Th16]. c-met polymorphism that is specific for papillary RCC [MYC pathway [The ACHN cell line represents an uncertain RCC histotype Fig. . It was lary RCC . Chromoslary RCC . Yet, ge pathway . No muta pathway .vhl [MYC analysis [A-498 is a “classical” RCC cell line belonging to the NCI-60 panel and is therefore widely used in cancer research. Mutated vhl , 47 woulvhl  detectedanalysis . Nonetheanalysis , 51 withanalysis .VHL expression, as it harbors mutated VHL [VHL+) cells are not significantly up-regulated in VHL mutated 786-O cells, confirming that the loss of VHL is not equivalent to hypoxic exposure and that in RCC, the VHL tumor suppressor has a distinct role from its activity in the hypoxia-inducible pathway [786-O, established as one of the first RCC cell lines, has many characteristics of ccRCC and is used most commonly in RCC-focused research Fig. . The 786ated VHL , 54 withated VHL  and giveated VHL , 56. In  pathway . Interes pathway , 59. Sur pathway  and vime pathway . These c pathway  as well, pathway , 63. A s pathway .vhl, it was shown to produce tumors of clear cell histology in nude mice, both by the total population and SP cells [Caki-1 is a widespread model line of metastatic ccRCC  in thosSP cells .VHL gene status of this cell line are inconsistent– some researchers detected no mutations [Caki-2 was established from a primary tumor of the kidney. This cell line was primarily defined as the ccRCC cell line Fig.  that exputations , while outations , 48 repoutations , 72, 73 utations –71, 74 rvhl and secretes high levels of VEGF, suggesting a ccRCC phenotype . Sixteen of the cell lines were derived from tumors composed predominantly of clear cell RCC, three were granular cell RCCs, and one papillary type (UOK112). The remaining tumors were of mixed types including clear and granular cells, clear cell + granular and sarcomatoid cells , or clear cell and papillary (OUK 120). Cell line phenotypes, including morphology, in vitro growth characterization, and tumorigenicity in nude mice were determined for these cell lines . AnotherSpecific subpopulations of cancer cells are available for culture. In particular cancer stem cells referred to as tumor-initiating cells are currently becoming available. In particular, donor specific kidney cancer cells derived from primary tumors cultured in stem cell-promoting media are enriched in CSCs . The role of CSCs in RCC has been reviewed elsewhere , as theyPrimary cultures of normal human epithelial cells of proximal origin derived from the renal cortex have been shown to present very homogeneous morphology in vitro . If normTotal kidney isolates are also available ; after the dissociation of a healthy renal sample, all cells are frozen which allows various cell types to be cultured without direct access to donors.Another widely used non-cancer renal cell line is the HEK293 cell line. Generated in 1977 by the viral transformation of human embryonal kidney cells,  HEK293 cRCC cell lines have also been developed as tools to study specific phenomena in RCC. In particular, a model to study the biology of the bone metastasis of renal cell carcinoma has been established. This cell line induced osteolytic lesions in nude mice after injecting into the tibia. RBM1 cell line cells expressed high levels of cytokines involved in osteoclast activation and bone resorption- parathyroid hormone-related peptide, interleukin-6, and macrophage colony-stimulating factor. Moreover, cells were confirmed to express EGFR and c-MET . SK-RC-4RCC subtypes less frequent than clear cell and papillary are even more difficult to study in vitro as specimens are obtained rarely. Recently, several cell lines were developed to model HLRCC; a rare genetic disorder that manifests by cutaneous and uterine leiomyomas and kidney tumors. Metastatic NCCFH1, UOK262, and primary UOK268 cell lines are FH deficient and can serve as models for hereditary papillary type 2 RCC , 130. Anvhl, pbrm1, bap1, setd2, kdm5c, pten and mtor as the most frequently mutated genes in ccRCC [vhl mutant cell lines are easily accessible; 786-O, 769-P, RCC4 (with reintroduced vhl control cell line available), are the most widely studied models for VHL role in RCC. Apart from gene mutation, vhl promoter region methylation often occurs in ccRCC samples which effects in no protein expression. Such a phenomenon was reported for SK-RC-54, 769-P, and A-704 [Some RCC cell lines, mostly present in NCI-60, were used in whole genome analyses. Mutational, copy variant, and expression analyses available for cells lines are provided as links to databases in Additional file in ccRCC . vhl mutnd A-704 .Classical cell lines were recently verified for PBRM1 expression status and confirmed ACHN- positive, 786-O– positive, and A-704– negative. The ACHN cell line expresses the protein, but harbors a heterozygous nonsense mutation, while 786-O, 769-P, Caki-1, and A-498 express wild-type PBRM1 , 134. ThBAP1 mutants are available as UM-RC-6, 769-P, and SN12C cell lines ; howevervhl, pbrm1, bap1, and setd2), 14q loss (hif1a), or 5q gain being the most frequent [Chromosome alterations are also common in ccRCC with the loss of the 3p chromosome . As mentioned above, ACHN cells have a MET polymorphism , but prmutation . This cemutation . A KDM6Amutation . A FAT1 Chromosome aberrations present in pRCC include chromosomes 7 and 17 gains and 9p loss . In the TP53 tumor-suppressor mutations, present in 50% of tumor cases in general, are less frequent in RCC (around 20% cases ), but coThe clinical translability of cell line experiments relies on valid animal models. They provide complex platforms for studying oncogenesis and the effectiveness of therapeutic approaches and, enable the verification of cell lines’ tumorigenicity. While tumorigenic cells can be implanted into the same species  or another species (xenografts), we will focus on immunocompromised experimental animals that are injected with human cell lines. The wide application of this approach in oncology studies followed the discovery of two groups of immunodeficient animals: “nude” mice  and latenu (Forkhead box protein N1) mutation, but more importantly they lack a thymus and are T-cell deficient but produce functional B-cells [scid) within the DNA-dependent protein kinase of the catalytic polypeptide Prkdc gene. This mutation affects both T and B lymphocytes [Athymic nude mice are hairless, an effect of the Foxn1 B-cells . This isphocytes . SCID rophocytes . Recentlphocytes . Still, The nature of the outcome of animal experiments strictly relies on the place and route of cell inoculation. Ectopic tumor xenograft models employ subcutaneous (s.c), intraperitenoeal (i.p.), intravenous (i.v.) or intramuscular (i.m.) implantation of tumor cells. While advantages are the approach’s simplicity and reproducibility, an obvious disadvantage of this approach is the non-physiologic growth location. An alternative approach, the orthotopic xenograft model, involves the implantation of a tumor into the originating tissue site of the cancer in rodents. In the case of RCC, the widely accepted implantation site is the renal subcapsule , 167. It3 in 2 weeks [Ectopic xenograft models of athymic nude mice with various genetic backgrounds have been extensively used for studying established RCC lines, including 769-P, 786-O, Caki-1, SK-RC-38, SK-RC-42, SK-RC-44, SK-RC-45, SK-RC-46, and others , 169–173 2 weeks . Implant 2 weeks . In addiGenerally, xenografted tumors are considered as not producing metastasis in most cases. Sharkey and Fogh  studied Finally, some cell lines fail to be tumorigenic in nude mouse; 769-P, SK-RC-7, TK 10, TK 164 UM-RC-6, or UOK108 , 179. TuDespite the undisputed profits that translational medicine has gained from research conducted on cell lines, several concerns have arisen regarding the extent to which cell line results can be trusted –183. DesCancer is a complex disease shaped by changes in cell functions, but also intrinsic signaling inside the tumor and extrinsic interactions with other cells of the host as well as various components of the local microenvironment. Such a multiplex disorder is very difficult to interpret, and cell line research does not always reflect cancer diversity. The limitations of the validity of cell line cultures apply also to RCC. To increase the usefulness of cell lines for RCC discovery, complex in vitro models have been designed. It was shown that samples from RCC patients cultured as 3D organoids create structures that closely mimic in vivo tumor, serving as a useful model for personalized drug screening . 3D cultWhen conducting research using established cell lines, one should carefully study the data on their establishment and subsequently available characteristics. Thorough data on original patients has not been was provided consistently in prior research; however, modern molecular analysis helps to characterize cell line features. Certain RCC specimens are underrepresented, such as: papillary and chromophobe RCC, metastatic ccRCC , and early stage tumors. When establishing new cell lines, it is essential to follow the best available guidelines, e. g. reviewed elsewhere . ThorougMolecular profiling of RCC cell lines is not always available, which may limit the clinical translation of in vitro research; back checking of model cell lines for typical RCC features is neededresearch on numerous cell lines with relation to healthy tissues can increase the clinical value of RCC cell line research; familiarity with their features is indispensable to draw accurate conclusionsAdditional RCC cell lines and culture models are needed to mirror in vivo heterogeneityWhen establishing new RCC cell lines thorough characterization of OP data and subsequent culture are indispensable to create a useful in vitro tool


Graft-versus-host response after allogeneic hematopoietic stem cell transplantation  represents one of the most intense inflammatory responses observed in humans. Host conditioning facilitates engraftment of donor cells, but the tissue injury caused from it primes the critical first steps in the development of acute graft-versus-host disease (GVHD). Tissue injuries release pro-inflammatory cytokines  through widespread stimulation of pattern recognition receptors (PRRs) by the release of danger stimuli, such as damage-associated molecular patterns (DAMPs) and pathogen-associated molecular patterns (PAMPs). DAMPs and PAMPs function as potent stimulators for host and donor-derived antigen presenting cells (APCs) that in turn activate and amplify the responses of alloreactive donor T cells. Emerging data also point towards a role for suppression of DAMP induced inflammation by the APCs and donor T cells in mitigating GVHD severity. In this review, we summarize the current understanding on the role of danger stimuli, such as the DAMPs and PAMPs, in GVHD. In addition, activated APCs produce an abundance of T-cell stimulating cytokines, such as IL-12, which further escalate the inflammatory response. In this review, we describe several encouraging investigations that have been conducted in both experimental bone marrow transplantation (BMT) models and humans over the last two decades. We further summarize the updated findings of how DAMPs and PAMPs amplify or mitigate GVHD and explore potential new strategies for the regulation of these “danger signals” in the regulation of GVHD.Allogeneic hematopoietic stem cell transplantation  has become widely used as a curative therapy for a variety of life-threatening hematological malignancies and congenital immune deficiencies . HoweverPAMPs are non-host derive molecules derived from microbes and are recognized by pattern recognition receptors (PRRs) that initiate and sustain the innate immune responses for protecting host from foreign pathogens . DAMPs aDanger signaling is transmitted through PRRs when they bind PAPMs and DAMPs. Several signaling pathways, such as toll-like receptor (TLR), Nucleotide-binding oligomerization domain (NOD)-like receptor (NLR), and retinoic acid-inducible gene 1 (RIG-I) signaling, are recognized. The detailed mechanisms are recently reviewed in several articles –7. In thToll-like receptors are one of the PRRs and play a key role in innate immune responses by recognizing PAMPs as well as DAMPs . TLRs ar−/− animals were used as either donor or recipient, acute GVHD severity and mortality were significantly ameliorated in MHC-mismatched B6 into BALB/c model by altering DC functions in TLR4−/− APCs  also showed significantly less intestinal GVHD, but reduction was appeared dependent on conditioning intensity . TLR4 signaling is transmitted through intracellular adaptor molecules myeloid differentiation primary response gene 88 (MyD88) and Toll/IL-1receptor (TIR)-domain-containing adaptor-inducing interferon β (TRIF) that activate NF-κB signaling that potently enhances expression of pro-inflammatory cytokines. The role of TLR4 in APCs for mediating acute GVHD remains controversial. Mutations in TLR4 are involved in the reduction of GVHD responses by hyporesponsiveness of APCs to LPS stimulation while over activation of TLR4 signaling results in exacerbation of GVHD . When TL−/− APCs . This fi−/− APCs . TLR2/4−ntensity . In minontensity . In addintensity , 15. Whi−/− animals were used as the host, acute GVHD was significantly improved (−/− T cell depleted BM (TCD-BM) cells showed greater intestinal GVHD (−/− donor T cells reduced graft-versus-tumor (GVT) activity through the expansion of Foxp3- and IL-4-producing T cells in MHC-mismatched haploidentical B6 into B6D2F1 model  from gram-positive bacteria as well as zymosan from yeast. Intriguingly, granulocyte-colony stimulating factor (G-CSF) mobilized donor grafts showed the increase level of TLR2 expression on myeloid cell populations , but uprute GVHD , 20.TLR5 recognizes flagellin that is an essential component of bacterial flagella from both gram-negative and -positive bacteria and regulates immunity –26. CBLBTLR7 recognizes endosomal single strand ribonucleic acids (RNAs) leading to production of type I interferons (IFNs), pro-inflammatory cytokines, as well as regulatory cytokines , 32. TLR−/− animals were used as hosts, acute GVHD severity and mortality was ameliorated. This was dependent on the expression of TLR9 expression on the non-hematopoietic cells (TLR9 recognizes unmethylated cytosine-phophorothionate-guanine (CpG) dinucleatides in the bacterial DNA and triggers a Th1-mediated inflammatory response . CpG-medic cells , 14. Howic cells , 43. Donic cells .−/− hematopoietic cells, we have already demonstrated that TLR3 deficiency in host APCs showed equivalent GVHD severity and mortality to WT animals but impaired GVT activity in MHC-matched multiple minor mismatched BMT model. Activation of TLR3 by polyinosine-polycytidylic acid (Poly I:C) improved GVT activity without enhancing GVHD (TLR3 recognizes double-stranded RNA (dsRNA), which is produced by most of viruses. It signals through interferon regulatory factor (IRF)3 and activates NF-κB and enhances production of type I IFNs , 46. TLRing GVHD . Becauseing GVHD , 50, TLR−/− recipient animals showed exacerbated GVHD severity and morality, particularly intestinal GVHD. This was due to host APC activation in experimental BMT  tripeptide from gram-negative bacterial PGN , 52, whintal BMT . By contMT model .+ T cells promotes IFN-γ production and Th1 differentiation by enhancing caspase 1-dependent IL-1β secretion mediated through intracellular C5 activation  (ASC), which has pyrin domain (PYD) and CARD, pro-caspase 1, and certain receptor proteins, such as NLR family members , the protein absent in melanoma 2 (AIM2) , 74. Onctivation . These dtivation . We recetivation , 84. Absallo-BMT . These rRIG-I, melanoma differentiation-associated gene 5 (MDA5), and laboratory of genetics and physiology 2 (LGP2) are known as RIG-I-like receptors (RLRs). RIG-I and MDA5 contain a DExD/H box RNA helicase domain and CARD, but LGP2 has no CARD-like domain. These receptors bind to intracellular dsRNA or ssRNA and trigger innate antiviral responses by producing type I IFNs –91. TherC-type lectin receptors (CLRs) are expressed on myeloid-derived APCs as soluble or transmembrane embedded proteins. They directly activate NF-κB through spleen tyrosine kinase (SYK)  or indirBoth exogenous and endogenous danger signal proteins are released from damaged tissues and abnormal intestinal microbial colonies after conditioning. In addition, blood stream infection (BSI) caused by gut translocation of colonized bacteria is another critical source of PAMPs after allo-HCT . ExperimLipopolysaccharides (endotoxin) are membrane component of many gram-negative bacteria representing one of the earliest and most investigated PAMPs in GVHD. The role of LPS in GVHD is complex and controversial. LPS translocation due to gastrointestinal (GI) tract damage is correlated with conditioning intensity  and show+ T cell and NK cell dependent model  that is stored within cytoplasm and mitochondria  as the primary energy source chondria . Once cechondria , 133. Inced GVHD . Anotherced GVHD . Increasced GVHD . In lineced GVHD . Loss ofted GVHD , 139. Thted GVHD . The immted GVHD .+ T cell cytotoxic functions, and is recognized as an endogenous DAMP (Uric acid is a metabolite of purine nucleotide and hyperuricemia is known to lead gout . Uric acous DAMP . Recent ous DAMP . Patientous DAMP . Howeverous DAMP .in vitro (in vivo effects on GVHD were not studied.Heat shock proteins (HSPs) work as molecular chaperones that enhance protein folding and intracellular transportation . HSPs hain vitro , but theHeparan sulfate proteoglycans (HSPGs) are component of extracellular matrix and play fundamental role in cell development, metabolism, and immunity , 157. HScandida species exacerbated GVHD in clinical studies  is a component of fungal cell wall as a known DAMP. The α-mannan is recognized by dectin1 and dectin2, which is one of the CLRs and activates NF-κB signaling through SYK and produces pro-inflammatory cytokines and effector T cell differentiation . α-manna studies , 101.“Danger signals” are indispensable role in initiating and developing acute GVHD. Regulating danger signal in an efficient manner in early phase of allo-SCT would ameliorate GVHD and have a great therapeutic strategy. Herein, we summarize potential therapeutic strategies for prevention and treatment of GVHD through modulating this signaling pathway. The studies are also summarized in Table P2X7 receptor on APCs binds to extracellular ATPs, which released from damaged tissues by conditioning, and activates APCs to produce pro-inflammatory cytokines. Therefore, P2X7R plays a key role in DAMPs-mediated inflammatory responses. Systemic administration of the broad-spectrum P2X7R antagonist, pyridoxal-phosphate-6-azophenyl-2′,4′-disulfonic acid (PPADS), or an ATP diphosphohydrolase, apyrase attenuated GVHD by suppressing APC activation . AdditioSerine protease inhibitor alpha-1 antitrypsin (AAT) attenuates GVHD by inhibiting HS, one of the DAMPs, and reducing HS mediated allogenic T cell responses in murine model . ClinicaSialic acid-binding immunoglobulin like lectins (Siglecs) have an immunoreceptor tyrosine-based inhibitory motifs (ITIM) or ITIM-like regions in their intracellular domains and negatively regulate DAMPs-mediated innate inflammatory responses , 178 or Danger signals mediate inflammatory responses through a multitude of PRRs that play a key role in the pathogenesis of GVHD. Once danger signals are released after conditioning, multiple innate immune signaling pathways are activated and amplified. Therefore, regulating danger signaling pathways in an effective manner is complex. Preclinical data suggest that targeting one specific signaling pathway or molecule may have only limited effects in reducing GVHD. In addition, the specific timing of regulation by blockade using antagonists may be a critical factor to consider. However, it is plausible that stimulating the negative regulating pathway that is commonly employed by several DAMPs may be a more rational way to mitigate GVHD. Thus exploring novel mechanisms of negative regulation of danger DAMP signaling that mediate lethal inflammatory responses should be carefully examined as new strategy of the prevention and treatment of GVHD. One potential benefit of regulating danger signaling is that GVT responses may be preserved due to selective attenuation of APCs with presumably limited effects on donor tumor-specific T cells necessary for mediating GVT responses. Clinical trials that investigate critical mediators of the danger response hold promise in the prevention of GVHD without affecting GVT responses.The review article was designed and written by TT and PR. Both NM and JM helped with designing figures. together with the help of co-authors NM and JM. All of them performed literature review and critically discussed the published literature.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


High-risk human papillomavirus (hrHPV) types induce immortalization of primary human epithelial cells. Previously we demonstrated that immortalization of human foreskin keratinocytes (HFKs) is HPV type dependent, as reflected by the presence or absence of a crisis period before reaching immortality. This study determined how the immortalization capacity of ten hrHPV types relates to DNA damage induction and overall genomic instability in HFKs.Twenty five cell cultures obtained by transduction of ten hrHPV types (i.e. HPV16/18/31/33/35/45/51/59/66/70 E6E7) in two or three HFK donors each were studied.All hrHPV-transduced HFKs showed an increased number of double strand DNA breaks compared to controls, without exhibiting significant differences between types. However, immortal descendants of HPV-transduced HFKs that underwent a prior crisis period (HPV45/51/59/66/70-transduced HFKs) showed significantly more chromosomal aberrations compared to those without crisis (HPV16/18/31/33/35-transduced HFKs). Notably, the hTERT locus at 5p was exclusively gained in cells with a history of crisis and coincided with increased expression. Chromothripsis was detected in one cell line in which multiple rearrangements within chromosome 8 resulted in a gain of MYC.in vitro, reflected by a crisis period, require more genetic host cell aberrations to facilitate immortalization than types that can immortalize without crisis. This may in part explain the observed differences in HPV-type prevalence in cervical cancers and emphasizes that changes in the host cell genome contribute to HPV-induced carcinogenesis.Together we demonstrated that upon HPV-induced immortalization, the number of chromosomal aberrations is inversely related to the viral immortalization capacity. We propose that hrHPV types with reduced immortalization capacity  A persistent infection with certain types of the human papillomavirus (HPV) has been causally associated with the development of cervical cancer, as well as a subset of other anogenital and head-and-neck cancers. Based on epidemiological data and functionally relevant genomic sequence differences, HPV types of the alpha-genus are classified as high-risk (hrHPV) , probable/possible hrHPV  and low-risk HPV  [in vitro via a two-step process, that involves the bypass of two mortality phases (M1 and M2) , such as gains at 1q and 3q . M1, oriewed in , 5. The iewed in , 7. Studiewed in –11. In tiewed by , 13).in vitro immortalization capacities and associated host cell aberrations differ amongst the various hrHPV types, we have recently established a large series of HPV-type specific in vitro models. Primary human foreskin keratinocytes (HFKs) isolated from two to three independent donors were transduced with the viral oncogenes E6 and E7 of HPV16, 18, 31, 33, 35, 45, 51, 52, 59, 66 and 70 [Despite the fact that HPV16 and HPV18 are the most common HPV types detected in cervical cancer, one third of cervical cancers can be attributed to other hrHPV types. To determine whether 6 and 70 . When st6 and 70 .This study aimed to determine whether HPV-type dependent immortalization capacities were related to differential DNA damage induction and copy number aberrations in immortal descendants. To this end, we analyzed HPV16-, 18-, 31-, 33-, 35-, 45-, 51-, 59-, 66- and 70-transduced HFKs, from two-to-three donors each, at distinct stages during immortalization, i.e. early passages representing pre-immortal cells in their extended lifespan and later passages representing immortal cell lines. Immortalization was previously determined by detection of telomerase activity and upregulated hTERT expression . In caseThe induction of DNA damage is a well-established feature of hrHPV types such as HPV16 and is known to contribute to HPV-induced cellular transformation . However, upon comparison between immortal cells that emerged from continuous growing ancestors and immortal cells that escaped from crisis, significantly more CNA were detected in the post-crisis cells Figure , which wA frequency plot for immortal cell lines with (n=9) and without n=11) a preceding crisis period is shown in Figure 1 a prece2 test, FDR<0.10) more often in immortal cells that underwent crisis, whereas loss of a region on chromosome 4p was significantly  more frequent in immortal cells showing continuous growth without an apparent crisis period  and without (n=11) crisis. Gains of two regions in chromosome 5p, 3 regions on 8q and a region on 9q as well as loss of two regions on chromosome 8p were observed significantly  and represents a precancerous lesion when cultured on organotypic rafts [One cell line  showed relatively few CNA genome-wide except for chromosome 8, with a strikingly high number of clustered breaks. Analysis after long-range paired-end sequencing revealed high number of intrachromosomal rearrangements on chromosome 8 and a single translocation connecting chromosome 4 with chromosome 8 Figure . Interesic rafts .In the present study we showed a progressive increase in chromosomal aberrations during hrHPV-mediated immortalization. Interestingly, we found that the frequency of aberrations was inversely related to the immortalization capacity of the ten hrHPV types analyzed. Cell lines immortalized by HPV types with a higher immortalization capacity, including HPV16, 18, 31, 33 and 35, showed significantly fewer CNA compared to the cell lines immortalized by HPV types with a lower immortalization capacity , in which immortalization was preceded by a crisis period. The increased number of CNA in cells that encountered a crisis period was not linked to a differential induction of DNA double strand breaks, as determined by γ-H2A.X staining, between HPV types with low and high immortalization capacities.In all hrHPV-transduced HFKs a significant increase in DNA-double strand breaks (γ-H2A.X positive cells) compared to controls was observed, with most cells being tested at early passage. Yet, most CNA became apparent at later passages following immortalization. This observation is in line with a previous report by Bester et al. in which DNA damage was immediately induced following expression of HPVE6 and E7 due to forced entry into S-phase without concomitant up-regulation of deoxyribonucleoside triphosphate synthesis, whereas loss of heterozygosity and copy number variations became evident after 100-250 population doublings . Since iCell lines without a crisis period and relatively few CNA were previously shown to express hTERT at early passages and to more efficiently degrade p53, indicating that the respective E6 proteins are more oncogenic than the E6 proteins of HPV types with reduced immortalization capacity . This suRegions on chromosomes 8q and 9q were more often gained in cell lines with a crisis period and may harbor (putative) oncogenes that provide a growth advantage in cells infected with less carcinogenic virus types during crisis. The lost region on chromosome 4p was significantly more frequent in cells that showed a continuous growth. Loss of heterozygosity at this region has been demonstrated in cervical carcinomas .in vitro transformed HPV-containing cell lines and may represent an effect of in vitro culturing [Overall, gain of chromosome 20q and loss of chromosome 3p and 22 represented the most frequent events in the HPV-immortalized cell lines . A gain ulturing , 35, 36.in vitro generated HPV-immortalized cell line with chromothripsis. Chromothripsis is characterized by massive rearrangements involving one or a few chromosomes and is a rare event that has been observed in 2 to 3% of all cancers [Interestingly and to the best of our knowledge, we are the first to report an  cancers . In one  cancers . Current cancers , these dOur preliminary data on mutation analysis of 48 cancer-related genes (TruSeq Amplicon panel Illumina) indicate that somatic mutations are extremely rare in HPV-immortalized cells. Only one mutation in GNAQ was found in one cell line. Furthermore, previous DNA methylation analysis of 15 genes in HPV16-, 18-, 31-, 33-, 45-, 66- and 70- immortalized cells showed no major differences in methylation patterns between the various HPV-immortalized cell lines [In conclusion, analysis of HPV-immortalized HFK cell lines transduced with ten different HPV types revealed that the induction of DNA-double strand breaks (phospho-histone H2A.X induction) is HPV-type independent. Conversely, the extent of CNA in immortal cells appeared to be HPV-type dependent. The percentage of CNA was found to be inversely related to the immortalization capacity of the virus type present. In particular the number of chromosomal gains was significantly increased in HPV-immortalized cells that underwent a crisis period compared to HPV-immortalized cells that grew continuously.in vitro immortalization capacity and apparent requirement of more genetic host cell aberrations to support an unlimited lifespan of HPV types 45, 51, 59, 66 and 70, compared to HPV types 16, 18, 31, 33 and 35, may in part explain their mostly lower prevalence in cervical cancers and precursor lesions.The reduced 2. Splitting dates and dilutions were noted to determine proliferation characteristics. Exponentially growing cells were harvested by trypsinization. Genomic DNA was isolated from cell pellets by proteinase K digestion followed by UltraPure™ Phenol:Chloroform:Isoamyl Alcohol (Life Technologies) extraction as described previously [Cell lines were retrieved and cultured as described before . Brieflyeviously .An overview of the various cell lines and passages used in the different experiments is given in Cells were grown in chamber slides (Nunc), washed with PBS and pre-permeabilized with 0.25% Triton X-100 in PBS (1 min on ice). Cells were fixed with 4% paraformaldehyde/PBS (15 min at room temperature) and permeabilized with 0.5% Triton X-100 in PBS (10–20 min at room temperature). Unspecific binding sites were blocked with 10% BSA/PBS for 1 h at room temperature. Slides were incubated for 2 h at room temperature with anti-phospho-Histone H2A.X  in blocking buffer and excess antibody was removed by four washing steps with 0.2% Triton X-100/PBS. Slides were then incubated with secondary antibody labeled with Alexa 488  and DAPI (1:1000) for 1 h at room temperature. Finally, slides were washed four times with 0.2% Triton X-100/PBS and embedded in vectashield . Slides were analyzed with a fluorescent microscope . Experiments were performed in duplicate and the percentage of phospho-histone H2A.X positive cells was scored from digitized images using constant light intensities. Approximately, 100-200 cells per experiment were counted and scored for γ-H2A.X positivity.http://www.ncbi.nlm.nih.gov/projects/geo/) through series accession number GSE72063.Labeling and hybridization to the 2×105K arrayCGH platform  was performed as described previously . Downstrhttp://www.ebi.ac.uk/ena; dataset ID: PRJEB9176.)Whole genome low-coverage short insert size library preparation was performed using the NEBNext Ultra DNA Library Prep Kit (New England Biolabs) and long-range (4422 bp insert size) mate-pair library preparation was done using the Nextera Mate Pair Sample Prep Kit (Illumina Inc.), both according to the manufacturer's protocol. Sequencing was performed on Illumina HiSeq instruments to an average 3x spanning coverage for short insert size paired-end and 38x spanning coverage for mate-pair, with the raw length of the reads displaying a median of 101bp. Structural variants larger than 5,000 bp were detected using the DELLY tool , as prev2-test as available in the R-package CGHtest was used to determine possible differences between chromosomal profiles of cell lines with a high immortalization capacity and cell lines with a low immortalization capacity [The χcapacity . The tes