Epithelial ovarian cancers (EOCs) are a heterogeneous group of tumors with different molecular and clinical features. In past decades, few improvements have been achieved in terms of EOC management and treatment efficacy, such that the 5-year survival rate of patients remained almost unchanged. A better characterization of EOCs’ heterogeneity is needed to identify cancer vulnerabilities, stratify patients and adopt proper therapies. The mechanical features of malignant cells are emerging as new biomarkers of cancer invasiveness and drug resistance that can further improve our knowledge of EOC biology and allow the identification of new molecular targets. In this study, we determined the inter and intra-mechanical heterogeneity of eight ovarian cancer cell lines and their association with tumor invasiveness and resistance to an anti-tumoral drug with cytoskeleton depolymerization activity (2c). Ovarian cancer (OC) is the seventh most common cancer and the fifth cause of cancer death among females . In 2018Despite the efforts in understanding EOC biology, the carcinogenesis of these tumors is poorly understood. Moreover, few improvements in patients’ survival have been achieved over the past 40 years due to late diagnosis, lack of effective treatments and tumor heterogeneity . A deepeIn recent years, cell biomechanics have been used for a more comprehensive characterization of EOCs’ heterogeneity both at the cellular and tissue level ,7. In 20In this regard, non-selective inhibitors of the deubiquitinating enzymes (iDUBs) are an interesting therapeutic option able to act on different cellular pathways. In particular, the DUB inhibitor 2c , developed in the laboratory of bio-organic chemistry from the University of Trieste , can triIn this study, we analyzed the mechanical properties of eight OC cell lines by atomic force microscopy (AFM) as characterization biomarkers of EOC inter and intra-mechanical heterogeneity. The same cell lines were submitted to an invasion assay and treated with 2c to test the association between cellular stiffness, metastatic potential and drug responsiveness. Lastly, the results of 2c treatment were validated by fluorescence staining for F-actin and β-tubulin in order to investigate the effects of 2c in the cytoskeletal organization.p = 0.0003)  .To characterize intra-tumor heterogeneity, in each cell line, the distribution of single-cell elastic moduli was analyzed, and the presence of different subpopulations was inferred by applying a peak deconvolution method ,14. The p < 0.0001). Moreover, TYKNU showed a Gaussian profile, while TYKNU CpR had a bimodal pattern where the mean Young’s modulus of the lowest-stiffness population (0.24 ± 0.12 kPa) was very close to that of TYKNU (0.28 ± 0.12 kPa).Notably, the average elastic modulus of TYKNU cell line was about two times lower than the corresponding cells resistant to cisplatin, namely TYKNU CpR   (p = 0.2)  . Howeverp: 0.05) . Overallp = 0.2) a.p = 0.0002; Morphologically, cell lines with fibroblastic-like morphology had a remarkably high number of invasive cells compared to those with epithelial morphology  were extracted from CCLE database and submitted to multiple Gene expression data obtained by ddPCR in all the cell lines investigated in the present study returned a higher expression level of AKT3 transcripts in bimodal cell lines, which were, in general, characterized by higher stiffness values .To evaluate the stiffness as a possible biomarker of response to 2c treatment, the IC50 value was measured in each ovarian cancer cell line and then related to the corresponding elastic modulus.p < 0.0001)  a. Accord2: 0.53, p: 0.04) , while the correlation was no more significant when considering only the lowest one , suggesting a possible implication of the stiffer population in the resistance to 2c  b. Notablce to 2c .No evident association, instead, was observed between the sensitivity to 2c and the morphological classification.Cells lines were split into two aliquots and treated with 20 µM of VV1 inactive drug control or 2c and after 7 h submitted to AFM. The mean Young moduli, before and after treatment, were recorded, and the coefficient of variation (CV%) of cell stiffness was calculated to investigate the drug effects on the mechanical proprieties of the cells. In addition, the IC50 and CV% parameters were dichotomized in “high” and “low” groups according to their median value (IC50 = 22 and CV% = 42) and used for further analyses.p = 0.0002; p = 0.03; 2: 0.55, p: 0.04)   c. Accord = 0.01) d.Concerning the effect of 2c on the distribution of single-cell elastic moduli, all cell lines with unimodal patterns retained a Gaussian profile after 2c treatment, while those with a bimodal pattern displayed a more heterogeneous behavior. In particular, HEY, OVCAR4 and TYKNU CpR continued to be non-normal after 2c administration, while SKOV3 and OVCAR8 lost their bimodality assuming a Gaussian curve. . OverallTo evaluate the 2c internalization and distribution inside the cell, OVCAR4 and HEY cells were treated with 2c-F2 (fluorescent label) and observed after 7 h. Those cell lines were chosen as representative of cells with the highest IC50 (HEY) and intermediate (OVCAR4). TYKNU cell line was excluded from this experiment because of the low IC50 value. The analysis of the fluorescence images showed that 2c-F2 was consistently incorporated at 6 h from administration . For botBased on the sensitivity to 2c, three cell lines, HEY, TYKNU and OVCAR4, were selected and submitted to immunofluorescence staining for F-actin to analyze stress fibers before and after drug administration. Accordingly, HEY cells had the highest IC50, TYKNU the lowest and OVCAR4 an intermediate value.Before treatment, the fluorescence intensity of F-actin was more concentrated in the cell center for the TYKNU cell line, the one presenting a unimodal distribution, low stiffness and IC50, while it was more diffuse throughout the cell in OVCAR4 and HEY, the cell lines characterized by a bimodal pattern, high stiffness and IC50. Notably, fiber organization was consistent with the cell line morphological classification: parallel bundles for the TYKNU and HEY (fibroblastic-like morphology) and tangled network for OVCAR4  .After 2c treatment, all three cell lines displayed disrupted actin cytoskeletons, with the accumulation of high-intensity spots and rings in the cell periphery. This effect was particularly evident for TYKNU and OVCAR4, while in HEY the intensity profile was more heterogeneous . The speTo further investigate the effects of 2c on cytoskeletal organization, immunofluorescence staining for β-tubulin was carried out in TYKNU, OVCAR4 and HEY before and after 2c treatment.Cell lines treated with the inactive form of 2c (VV1) showed a “bush” organization characterized by microtubule filaments from the cell center to the periphery. OVCAR4 displayed higher intensity in the cell center compared to the border, while HEY and TYKNU presented a more diffuse pattern all over the cell.On the contrary, after treatment with 2c, cells presented a sharp splitting of the microtubule network, which appeared disorganized in filaments throughout the cell. In particular, the fluorescence intensity of TYKNU cells was higher in the cell center compared to the border, while in OVCAR4 and HEY it was prominent on the cell periphery . The spaIn this study, AFM was applied to investigate the inter and intra-mechanical heterogeneity of eight EOC cell lines and their associations with tumor invasiveness and response to 2c treatment. Tumor heterogeneity was inferred by applying a fitting deconvolution method on single-cell stiffness distribution. The resulting curves showed mainly two different patterns: a unimodal distribution in three cell lines and a bimodal pattern in five. The latter was characterized by two main peaks indicative of two populations with different mechanical features.Intra-mechanical heterogeneity can be an important marker of drug sensitivity.Our results on the IC50 analysis for 2c have indeed shown that cell lines with a bimodal pattern are more resistant to treatment than those with a unimodal pattern. This was particularly evident for TYKNU and its cisplatin-resistant subline, TYKNU CpR, which were, respectively, unimodal and bimodal. In addition, TYKNU CpR was significantly stiffer compared to TYKNU. This is in agreement with Raudenska et al., who reported an enhancement in cellular stiffness mediated by cisplatin treatment in prostate cancer cell lines . NotablyIntra-mechanical heterogeneity of cell lines has already been correlated with drug sensitivity in triple-negative breast cancer cell lines by optical stretching . By a maIn ovarian cancer lines SKOV3 and OVCAR5, Sharma and co-authors reported a bimodal distribution of the Young modulus by AFM in the cis-platinum resistant replicates, namely OVCAR5-CisR and SKOV3-CisR. In agreement with us, the stiffness of the softer subpopulation in the resistant subline was similar to the corresponding sensitive ones .Despite that several studies have indicated that low stiffness was associated with higher tumor invasiveness ,18,19, oIn our study, clinical data of the patients from which the eight EOC cell lines were isolated further support the potential of intra-mechanical heterogeneity as a marker of drug responsiveness. Cell lines with a bimodal pattern were indeed derived from patients with platinum or Adriamycin resistance, while three out of four cell lines with unimodal profiles were obtained from patients with no reported drug resistance ,26,27,28The 2c is a non-selective and irreversible inhibitor of isopeptidases of possible use in tumor treatment . Its priOur results showed that 2c administration led generally to a significant decrease in cell stiffness, which well fits with its action on the depolymerization of the F-actin network by activation of Cofilin-1 . Those rFibroblastic-like cell lines were characterized by a remarkable higher number of invasive cells than those with epithelial morphology. It is well known that cancer invasion ability depends also on the degree of epithelial differentiation within the tumor: poorly differentiated tumors are usually more invasive than well-differentiated ones. For this reason, cancer cells need to undergo an epithelial–mesenchymal transition (EMT) to lose adhesion and epithelial morphology and to assume, in this way, mobility with a mesenchymal–fibroblast morphology . AccordiTaken this evidence, our results on invasion assay in function of morphological classification are in agreement with the fundamental processes underlining the metastatic diffusion.Cell lines with unimodal and bimodal stiffness distribution seem to modulate differently their elastic proprieties after 2c administration. Our results indicated a tight association between the stiffness distribution pattern , the CV% of cellular stiffness after 2c treatment and the IC50 value. The CV% was remarkably lower in cell lines with higher resistance to 2c compared to the sensitive ones. Therefore, the softer the cells are after treatment the higher the response. In agreement with this, we found that cell lines with bimodal pattern, which displayed a lower CV% compared to the unimodal ones, were also more resistant to 2c. Results from RNAseq analysis highlighted a significantly higher expression of genes involved in cellular stiffening, 2c response and EMT in EOC cell lines with bimodal pattern as reported in ovarian cancers as well as in other cancer types during progression ,43,44,45Overall, this study highlights that intra-mechanical heterogeneity can be a possible marker for 2c response and cell invasiveness. We acknowledge however that our results are based solely on cell line models; therefore, they should be validated in a broader number of cell lines/primary cell culture as well as with in vivo experiments allowing a deepening of tumor features for clinically relevant conclusions.Eight epithelial ovarian cancer cell lines were analyzed in the present study. SKOV3 and IGROV1 cell lines were generously provided by Prof. G. Ricci , while HEY, OVCAR8, OVCAR4, TYKNU, TYKNU CpR and OAW42 cell lines were provided by Prof. O.M. Carpen . The TYKNU CpR is a cis-platinum resistance cell line derived from the treatment of TYKNU cell line with a stepwise concentration of cis-platinum .2 and were plated in filtered cap flasks . To discriminate between the fibroblastic-like and epithelial-like morphology the “Aspect ratio” and “Circularity” of cells were investigated as already described [The HEY, OVCAR4 and OVCAR8 were cultured in RPMI 1640 medium  supplemented with 10% fetal bovine serum , 1% Streptomycin-Penicillin 25×  and 1% Gentamicin . The SKOV3 and OAW42 were grown in high-glucose DMEM medium  supplemented with 10% FBS, 1% streptomycin-Penicillin 25× and 1% Gentamicin. Finally, the TYKNU and TYKNU CpR cell lines were cultured in EMEM medium  supplemented with 10% FBS, 1% streptomycin-Penicillin 25× and 1% Gentamicin, while the IGROV1 cells were grown in DMEM: F12 (1:1) medium supplemented  with 10% FBS, 1% streptomycin-Penicillin 25× and 1% Gentamicin. All cell lines were incubated at 37 °C in a humidified atmosphere of 5% COescribed . Cells wFor each cell line, AFM measurements were carried out in about 100 cells in three different experiments. For AFM measurements an MF3D-Bio AFM  with an inverted optical microscope  was used. The mechanical measurements were performed using the cantilevers PNP-TR-TL-50  with a triangular shape and nominal elastic constant of 0.32–0.08 N/m. The cantilevers were functionalized with silica beads with a diameter of 4.5 μM to allow cell microindentation and decrease simultaneously the risks of sample damage. As the first step, cells were seeded on a glass coverslip and analyzed by AFM within 2–3 days from seeding. Measurements were carried out in complete medium warmed at 37 °C to keep cells healthy during the analysis. For each analyzed cell at least five force-displacement curves at different locations in the cell peri-nuclear region were collected. The microindentation was performed at an indentation velocity of 2 μm/s, slow enough to avoid hydrodynamic effects, and a force-distance of 5 μm/s, large enough to ensure the tip will be fully detached from the cell between indentation instances. Moreover, the deflection trigger point was settled at 2 nN to avoid cell damage . The rec6 cells/mL was injected into the cell culture insert. After 72 h of incubation, the non-invading cells that had not moved to the underlying well were washed out from the insert, while invading ones were stained and counted through an optical microscope following manufacturer instructions. The average invading cell number was achieved by cell count of at least three optical fields at 10× magnification.The metastatic potential of the cell lines was assessed using a cell culture insert . For each cell line, a cell suspension aliquot in serum-free medium containing 0.9 × 10http://sites.broadinstitute.org/ccle/ (accessed on 4 April 2023) for 6 out 8 cell lines, namely IGROV1, OAW42, OVCAR8, OVCAR4, SKOV3, TYKNU. In detail, genes involved in cytoskeleton organization, cell cycle control, epithelial mesenchymal transition, invasiveness and response markers to 2c were exported and analyzed. Overall, 80 gene profiles were analyzed , the expression analysis was performed by RT-ddPCR using 1 ng of cDNA. RT-ddPCR analyses were carried out as already reported , with thEach cell line was treated for 24 h with increasing concentrations of 2c: 1–2–4–10–20–40–80 μM . OVCAR4, HEY and TYKNU cells were split into two populations for the treatment with 20 μM 2c and VV1  was used at a concentration of 2 μg/mL for 45 min to detect the primary antibody/β-tubulin complex. After staining with DAPI, the coverslips were assembled on microscope slides and left at 4 °C overnight. Finally, cell line images were taken at the fluorescence microscope (Leica DM2000) at a magnification of 20× and 40× and analyzed by ImageJ 1.53j.As concerns the microtubule network, an immunofluorescence assay for β-tubulin was carried out. Similarly to actin network analysis after splitting cells into two populations for treatment with 2c and VV1, they were fixed after 24 h in 4% paraformaldehyde and 0.15% picric acid. After treatment with 0.1% Triton X 100 and 1% BSA for membrane permeation, fixed cells were treated with the primary antibody for β-tubulin at a concentration of 10 ng/mL for one hour at room temperature. Subsequently, an anti-rabbit secondary antibody labeled with a green fluorophore , R 4.04 and Origin 2021 .Data were expressed as mean ± standard deviation. To assess the statistical relevance, the comparisons between groups were performed using parametric or non-parametric test according to variables distribution. Pairwise correlation analyses were carried out using Pearson correlation, while linearity was tested by regression analysis. Data distribution was assessed by the Shapiro–Wilk test. Continuous variables such as IC50, invasiveness score and CV% were dichotomized in “high” and “low” groups according to their median value. The association between categorical variables was assessed by Pearson’s chi-squared test. The fluorescence intensity was reported as the mean pixel intensity (m.p.i.), which is the sum of the color intensity values of all pixels in the selected area divided by the number of pixels. All


Unlike normal cells, cancer cells frequently have multiple centrosomes that can cluster to form bipolar mitotic spindles and allow for successful cell division. Inhibiting centrosome clustering, therefore, holds therapeutic promise to promote cancer cell‐specific cell death.We used confocal microscopy, real‐time PCR, siRNA knockdown, and western blot to analyze centrosome clustering and declustering using normal lung bronchial epithelial and nonsmall‐cell lung cancer (NSCLC) cell lines. Also, we used Ingenuity Pathway Analysis software to identify novel pathways associated with centrosome clustering.In this study, we found that exposure to cigarette smoke condensate induces centrosome amplification and clustering in human lung epithelial cells. We observed a similar increase in centrosome amplification and clustering in unexposed NSCLC cell lines which may suggest a common underlying mechanism for lung carcinogenesis. We identified a cyclin D2‐mediated centrosome clustering pathway that involves a sonic hedgehog‐forkhead box protein M1 axis which is critical for mitosis. We also observed that cyclin D2 knockdown induced multipolar mitotic spindles that could eventually lead to cell death.Here we report a novel role of cyclin D2 in the regulation of centrosome clustering, which could allow the identification of tumors sensitive to cyclin D2 inhibitors. Our data reveal a pathway that can be targeted to inhibit centrosome clustering by interfering with the expression of cyclin D2‐associated genes. We performed Real‐Time PCR, siRNA knockdown, western blot, and confocal microscopy to analyze centrosome clustering and declustering using normal lung bronchial epithelial cells exposed to CSC and compared to non‐small cell lung cancer (NSCLC) cell lines. We identified a cyclin D2‐mediated centrosome clustering pathway that involves SHH‐FOXM1‐CCND2‐CDK2 axis which is critical for mitosis plays a key role in CSC induced Beas‐2B cells. This study suggests a novel role for cyclin D2 in the regulation of centrosome clustering and reveal a pathway that can be targeted to inhibit centrosome clustering by interfering with cyclin D2 associated genes. Cigarette smoke induces genomic instability through a well‐understood general mechanism, however, alterations in the centrosome dynamics are not known. The generation of more than two centrosomes, termed supernumerary centrosomes, is common in human cancers and correlates with chromosomal instability.Drosophila melanogasterSeveral studies have reported that inducing centrosome amplification increases tumor formation in We have previously reported that two major carcinogens present in cigarette smoke, Benzo[a]pyrene (BaP) and 4‐(methylnitrosamino)‐1‐(3‐pyridyl)‐1‐butanone (NNK), perturb the mitotic spindle apparatus and induce centrosome amplification in human lung epithelial cells.22.12 atmosphere, which were incubated until reaching 80% confluence.Beas‐2B human bronchial epithelial cells, two lung adenocarcinoma cell lines (H23 and HCC827), one squamous lung cancer cell line, H1703, and a large cell lung carcinoma cell line (H460) were obtained from ATCC® . The lung adeno, squamous, and large cell carcinoma cell lines were maintained in an RPMI‐1640 medium according to the recommended protocol. Beas‐2B cells were cultured as described in our previous study.2.24 cells/well in a 96‐well plate. Different percentages of CSC  were added to the medium for 72 h. Next, 50 μl of XTT labeling mixture was added to each well . Optical density was measured in triplicate wells at 490 nm with a reference wavelength of 650 nm using a Biotech® microplate reader.Beas‐2B cells were seeded at 1 × 102.3In the smoke extraction preparation, cigarettes were burned for 4–5 min/cigarette to mimic smoke exposure. For the preparation of CSC media, a burned cigarette was bubbled through a 100 ml BEBM medium in a special chamber. The resulting medium was termed 100% CSC cell culture media. After obtaining results from the XTT assay where cell viability with 10% CSC was 90% , 25 mM HEPES, 0.05% glutaraldehyde, 10 mM EGTA, and 4 mM MgSO4, was used to fix cells for 10 min. After washing in PBS, cells were blocked with 2.5% normal goat serum for 15 min, then incubated overnight with primary antibody (1:1000) at 4°C. After washing with PBS, cells were incubated with 1:500 Alexa Fluor‐conjugated secondary antibody at room temperature for 2 h. For each pair of primary–secondary antibodies, the same steps were sequentially repeated.Immunofluorescent staining was conducted using standard protocols.Primary antibodies used include Rabbit polyclonal antipericentrin (Abcam® Cat. no. ab 4448) and mouse monoclonal anti‐α‐Tubulin (Sigma® Cat. no. T‐9026). Secondary antibodies used include Alexa Fluor 488 or 555 (Invitrogen®). Nuclei were stained by incubating cells with mounting media containing DAPI .2.6Fluorescence imaging was conducted on the Nikon A1R® confocal imaging system equipped with a Nikon® oil immersion Plan‐APO 60x numerical aperture 1.40 lens. The Nikon® NIS Elements software was used for imaging control and projections. With acquisition settings kept constant, Z‐stacks of images at 0.2‐mm intervals were acquired, and maximum intensity projections were created.2.7As centrosome clustering may be very tight, counting exact centrosome numbers in each cluster is difficult. An acceptable alternative for measuring centrosome clustering is measuring the volume of each centrosome cluster using a published protocol.2.8Metaphase, anaphase, and telophase cells were scored for the presence or absence of lagging chromosomes and bridges . A total of 50 cells were scored to determine the chromosome segregation errors and a two‐tailed Fisher exact test was used to analyze group differences.2.9To identify genes associated with centrosome clustering, we used a custom‐designed plate with mitotic spindle‐associated genes2.10To assess protein expression, western blot analysis was conducted for selected genes identified in our RT‐PCR analysis. Equal amounts of protein (50 μg) from each cell lysate sample were separated by SDS‐PAGE and transferred onto a PVDF membrane by electroblotting (Bio‐Rad). All primary and secondary antibodies used in the western blot analysis were purchased from Abcam®. Membranes were exposed to X‐ray film after incubation with enhanced chemiluminescence. Western blots shown are representative of at least three replicates.2.11To silence CCND2, SHH, and FOXM1, Beas‐2B cells were transiently transfected with SMARTpool siRNA  using the transfection reagent Lipofectamine according to the manufacturer's instructions . siRNAs‐targeting CCND2 (E‐003211‐00‐0020), SHH (E‐006036‐00‐0020), and FOXM1 (L‐009762‐00‐0020) as well as nonspecific control siRNA were used.2.12p value). The z‐score measures how well the observed and predicted patterns of up‐ and downregulation match.We used ingenuity pathway analysis (IPA) network analysis (Qiagen) based on our previously published study2.13p value of <0.05.Prism 7 (GraphPad® Software Inc.) was used for statistical analyses. CSC‐treated cells, untreated control cells, and NSCLC (adeno and squamous cell carcinoma) cell lines were compared using two‐way ANOVA analysis. The significance of differences between cell lines was assessed with Bonferroni's multiple comparison test, with differences considered significant with a 33.150 value. Figure 50 of 16.9%, with over 90% cell viability at 10% CSC concentration thus allowing for the accurate evaluation of mitotic alterations.We have previously reported that BaP and NNK induce mitotic abnormalities such as centrosome amplification, misoriented spindles, and misaligned chromosomes at metaphase.3.2p < 0.0001). We determined centrosomes volumes by measuring pericentrin intensity using Nikon Elements 5.1 software according to our previously published procedure , it was considered centrosome amplification. CSC‐exposed Beas‐2B cells showed an increased volume of centrosome compared with vehicle‐treated control  in response to CSC   Figure  in cance3.3Since similar genes were upregulated in CSC‐treated Beas‐2B and vehicle‐treated human NSCLC cell lines (two adenocarcinomas and one squamous cell carcinoma), we analyzed the expression of 20 mitotic genes by RT‐PCR analysis in these cell lines to identify genes that play a key role in centrosome clustering. We observed a marked increase of CCND2, FOXM1, and SHH (>2‐fold) in both CSC‐treated Beas‐2B and NSCLC cell lines Figure . The rem3.4p < 0.0001). This suggests that interfering with CCND2 could disrupt the centrosome clustering mechanism.As we observed CCND2, SHH, and FOXM1 overexpression in response to CSC and a similar or higher level in NSCLC cancer cell lines, we, therefore, treated Beas‐2B cells with short interfering RNAs (siRNAs) for all the three genes in order to determine the effect of silencing these genes in centrosome clustering. The siRNA knockdown was confirmed by western blot analysis Figure . We foun3.5We observed CCND2 overexpression in both CSC‐treated Beas‐2B cells and NSCLC cell lines, and studies have shown overexpression of CCND2 in gastric cancer that is associated with tumor progression and metastasis.4In the present study, we report centrosome clustering in human lung epithelial cells in response to cigarette smoke, for the first time to our knowledge. We have identified a pathway involving SHH‐FOXM1‐CCND2 which was elevated in response to exposure to CSC, and the genes associated with this pathway could play a role in centrosome clustering in NSCLC cell lines. Our previous studies showed that NNK and BaP, two major carcinogens present in cigarette smoke, perturb the mitotic spindle apparatus and induce centrosome amplification in lung epithelial cells.The presence of multiple centrosomes in a cell, known as centrosome amplification, has been accepted as a hallmark of cancer. These supernumerary centrosomes can cause a mitotic catastrophe,In addition, we also observed other genes that were differently expressed in NSCLC cells Figure . We idenDisrupting the clustering of centrosomes is thought to have great potential as a therapeutic strategy.This study shows for the first time a CSC‐induced centrosome clustering pathway representing a novel mechanism by which cancer cells may progress through the cell cycle and survive with continued proliferation. CCND2 overexpression may have a key role in the development of NSCLC and inhibiting CCND2 could be a novel therapeutic strategy to suppress the proliferation and survival of lung tumor. Previous studies show that CDK2 is the major conductor of the centrosome cycle and that it mediates centrosome amplification.Jose Thaiparambil: Conceptualization (lead); data curation (lead); formal analysis (lead); investigation (lead); methodology (lead); project administration (lead); resources (lead); software (lead); supervision (lead); validation (lead); visualization (lead); writing – original draft (lead); writing – review and editing (lead). Chandra Sekhar Amara: Methodology (supporting); writing – review and editing (supporting). Subrata Sen: Methodology (supporting); writing – review and editing (supporting). Nagireddy Putluri: Methodology (supporting); writing – review and editing (supporting). Randa El‐Zein: Conceptualization (lead); data curation (lead); formal analysis (lead); funding acquisition (lead); investigation (lead); project administration (lead); resources (lead); supervision (lead); visualization (lead); writing – review and editing (lead).This research was supported by the NIH/NCI U01CA189240 (REZ), NIH/NCI R01CA216426 (N.P./REZ), NIH/NCI R01CA220297 (N.P), DOD W81XWH‐21‐1‐0613 (N.P), P30ES030285, and P42ES027725.The authors declare that they have no conflict of interest.No human or animal studies are presented in this manuscript. No potentially identifiable human images or data are included in this study.Figure S1Click here for additional data file.Figure S2Click here for additional data file.


VRK1 and MAN1A2 were increased by several drugs in both cell lines. However, they display opposite effects, circ/linVRK1 favors apoptosis whereas circ/linMAN1A2 stimulates cell migration, and only XL765 did not alter the ratio of other dangerous circ/linRNAs in MCF-7. In MDA-MB-231 cells, AMG511 and GSK1070916 decreased circGFRA1, as a good response to drugs. Furthermore, some circRNAs might be associated with specific mutated pathways, such as the PI3K/AKT in MCF-7 cells with circ/linHIPK3 correlating to cancer progression and drug-resistance, or NHEJ DNA repair pathway in TP-53 mutated MDA-MB-231 cells.Altered expression of circular RNAs (circRNAs) has previously been investigated in breast cancer. However, little is known about the effects of drugs on their regulation and relationship with the cognate linear transcript (linRNA). We analyzed the dysregulation of both 12 cancer-related circRNAs and their linRNAs in two breast cancer cell lines undergoing various treatments. We selected 14 well-known anticancer agents affecting different cellular pathways and examined their impact. Upon drug exposure circRNA/linRNA expression ratios increased, as a result of the downregulation of linRNA and upregulation of circRNA within the same gene. In this study, we highlighted the relevance of identifying the drug-regulated circ/linRNAs according to their oncogenic or anticancer role. Interestingly,  CircRNAs, either non-coding or coding RNA molecules, can act as oncogenes or tumor-suppressors, and might be useful in early diagnosis or as novel targets for therapeutic approaches ,2. The i+ cells .− and basal-like subtypes. In contrast, low pAKT levels are generally present in luminal A tumors (and MCF-7 cells) [We focused on BC using two widely adopted cell lines, MCF-7 and MDA-MB-231, representing different subtypes, in which PI3K/AKT1/mTOR axis is one of the most affected. In fact, the mutation/loss/duplication in genes within this pathway correlates with poor prognosis, and higher Akt activation, mainly in the HER27 cells) . Thus, w7 cells) , such asTherefore, we studied the effect of 14 anticancer drugs  on 12 ciGenerally, dysregulated gene expression is studied in cancer upon drug stimulation without considering alternative transcript forms, such as circRNAs. For this reason, we aim to investigate the levels of circRNAs compared to their linRNAs, pointing out pharmacological agents that could interfere with this type of aberrant splicing. Focusing on circular/linear deregulation, we considered 12 circRNAs that emerge in the literature as modulated in tumor tissues or cell lines with disclosed functional roles in BC. They are listed in We first investigated the impact of the 14 compounds reported in −ΔCT formula and Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as the reference gene. We performed a statistical analysis using both the t-test (two-tail paired) and the Benjamini–Hochberg (BH) correction to assess the changes in the expression of circRNAs and linRNAs by each drug. In 50 value, specific for each cell line, as reported on the website https://www.cancerrxgene.org (accessed on 17 January 2023). From the data analysis in p-values < 0.05, calculated using the BH correction, while AZD7762, GEFITINIB, DOXORUBICIN dysregulated mostly linRNAs. Finally, XL765 modified the expression of both types of transcripts. In MDA-MB-231 cells, instead, only AZD5363 changed the levels of circRNAs, while AZD7762, ERLOTINIB, GEFITINIB, GSK1070916, and DOXORUBICIN altered linRNAs’ expression. We can resume that in both cell lines AZD5363 interfered with the circRNA expression, whereas the compounds AZD7762, GEFITINIB, and DOXORUBICIN interfered preferentially with linRNA transcription. Other drugs seemed to act in a more specific manner. On the one hand, AZD8055 and DOCETAXEL drove the dysregulation of circRNAs in MCF-7 cells, including XL765, which also altered the linRNAs; on the other hand, ERLOTINIB and GSK1070916 modified linRNAs in MDA-MB-231 cells.CircRNAs levels have been quantified by Reverse Transcription and quantitative polymerase chain reaction (RT-qPCR) both in MCF-7 and MDA-231 cells treated with the drugs. Four to six independent experiments were carried out in duplicate, and the expression of each circRNA and cognate linRNA has been quantified by calculating the fold change (FC) value using the 2AKT2 was the only unaffected transcript. We evidenced that LEE011-modulated circNCOA3 is the only one with a statistically significant adj p-value.Focusing on a single cell line, in MCF-7 we identified eight circRNAs  and 8 linRNAs  differentially expressed after exposure to drugs, quantifying the FC with respect to the untreated cells . GSK1070We underlined that DOCETAXEL, ERLOTINIB, and LEE011 impacted circRNAs and not the linRNAs, decreasing their expression; conversely, AZD7762 and GEFITINIB did not affect circRNAs, but interfered with the expression of linRNAs, again reducing their levels.p-value < 0.05), XL765 modified all eight circRNAs, followed by AZD5363 modifying six of them, DOCETAXEL deregulating five, and AZD8055 and DOXORUBICIN changing the levels of four circRNAs. While these drugs impacted a large number of circRNAs, others altered a specific circRNA, for instance BYL719, affecting circHIPK3, ERLOTINIB with circ000911, and LEE011 dysregulating circNCOA3.Among the analyzed compounds , among them, three were altered by DOXORUBICIN, two by AZD5363, AZD7762, and LEE011, while circGFRA1 was modulated exclusively by AMG511 and GSK1070916. The same drugs also modulated most of the linRNAs. Ten linear transcripts  changed their expression after treatment, eight of them with AZD7762 and six with DOXORUBICIN, while five linRNAs changed with GSK1070916, four with AZD5363, three with ERLOTINIB and XL765; finally, GEFITINIB influenced two linRNAs and AZD8055 only linMAN1A2.AKT3 gene, AZD7762 with NCOA3 and VRK1, DOXORUBICIN with MAN1A2 and NCOA3.Three molecules were able to modify the expression of both the circRNAs and their cognate linRNAs: AZD5363 associated with the p = 0.001). As shown in VRK1 and NCOA3, while the others, GFRA1 and MAN1A2 were selectively modulated by AZD5363 and AZD8055, respectively.To clarify whether a drug differentially interfered with the expression of the circRNA and cognate linRNA, we compared the “FC circRNA/FC linRNA” ratio. We considered those significantly dysregulated . Thus, wp = 0.013). AZD5363 altered the expression of other genes in MDA-MB-231 (MAN1A2 and AKT2) compared to MCF-7 cells .A similar analysis, carried out on MDA-MB-231 cells , showed + subtype. We focused on the HER2− cell lines, in order to perform a robust statistical analysis, rather than dispersed in a shallow study.This study would broaden the concept of biomarkers ,23, applFrom the evaluation of each specific RNA/drug interaction, it can be highlighted that the drugs globally impacted on circRNAs and linRNAs of the host gene, leading to a reduction of linear transcripts and a gain of the circular forms. This behavior has been reported recently following treatment with Actinomycin D, which blocks transcription and was explained as a consequence of higher stability by the circRNA . NeverthIt is important to underline that the circRNA and the cognate linRNA may have opposite functions. Indeed, while some circular and/or linear RNAs among those we studied might inhibit processes that support tumorigenicity, others implemented drug resistance or cell motility. For this reason, we highlight the importance of the drugs that modify a single circular or linear RNA in a specific type of cell. BYL719 to circHIPK3, ERLOTINIB to circ000911 and LEE011 to circNCOA3 in MCF-7 , while AConsidering that increased circHIPK3 levels represent a negative event often associated with the development of drug resistance , so muchConcerning the correlation of drug-mediated activity on a linRNA, GEFITINIB modified the expression of linSNX27, in MCF-7 , a promoMAN1A2 and VRK1 genes were dysregulated in both cell lines by different drugs. CircMAN1A1 is a marker of active cell proliferation in several types of tumors [Focusing on the ratio of the circRNA/linRNA pairs, the treatment with AZD5363 affected both cell lines, whereas AZD8055 and BYL719 were selective for MCF-7  and AZD7f tumors ,30, and f tumors . We detef tumors . It is rNCOA3, an oncogene [NCOA3 polymorphisms are associated with the risk of the development of BC [Instead, oncogene , was dysnt of BC .We point out that it is crucial to identify what are the circRNAs and cognate linRNAs modulated by anticancer agents concerning their putative functions and cell-specific mutated pathways. We summarized the pathways affected by the investigated drugs associated with the modulated circRNAs or linRNAs emerging by our data in Analyzing these data in relationship with the key mutations detected in MCF-7 and MDA-MB-231 cells  [BCL11B, whose decrease might display a role as an inhibitor of cell differentiation [SXN27. The latter is involved in the MAPK signaling targeted by ERLOTINIB and GEFITINIB.About the PI3K/Akt pathway, the linear isoform of the  therapy , decreas therapy , was als therapy , and othnd ATF2) ,45,46,47nd ATF2) , interfend ATF2) . Althougnd ATF2) , as wellnd ATF2) . In MDA-ntiation , as wellTaken together, these data suggest that circRNAs and linRNAs dysregulation could be associated with gene mutations affecting selective pathways as revealed by specific inhibitors of driver genes.Finally, we can mention that the biogenesis of circRNAs is strongly associated with epigenetic modifications, such as the H3K36me3specific histone mark , which d+/PR+/HER2−) and the triple-negative MDA-MB-231 (ER−/PR−/HER2−), purchased from the American Type Culture Collection . They were cultured in Dulbecco’s Modified Eagle’s Medium  supplemented with 10% fetal bovine serum , 2 mM L-Glutamine  and 50 U/mL penicillin plus 50 µg/mL streptomycin . The cells were grown at 37 °C in a 5% CO2 humidified atmosphere. MCF-7 were plated in T25 flasks at an approximate density of 6,4 × 105 cells/cm2 and MDA-MB-231 cells at 3,6 × 105 cells/cm2 and exposed to 14 different drugs purchased from Chemietek  for 24 h, already described by Baldassari et al. [50 concentration, or 1 μM (for the compounds with IC50 higher than 1 μM), and AMG511 at 60 nM. The IC50 for most of the used compounds reported in cancerrxgene  is also indicated in For this study, we have employed two human BC cell lines, the hormone-responsive luminal A MCF-7 .These cells were characterized by specific mutations, the most important were reported in ® Reagent , following the instruction provided by the supplier. Purified RNA was quantified with NanoDrop™ 1000 Spectrophotometer  and 2 µg reverse transcribed using SuperScript® II Reverse Transcriptase  using the protocol from Thermo Fisher Scientific.We carried out total RNA extraction from untreated cell samples or after drug treatments using TRIzolhttps://www.bio-inf.cn/ accessed on 31 January 2022) [https://www.ncbi.nlm.nih.gov/tools/primer-blast/ accessed on 31 January 2022), to generate the primers to amplify the linRNAs, purchased from Merck. The reactions were carried out using Thermal cycler CFX96 Touch™ Real-Time PCR Detection Systems  for 40 cycles, as follows: (i) enzyme activation: 95 °C for 3 min; (ii) denaturation: 95 °C for 20 s; (iii) annealing and extension as reported in GAPDH was used as endogenous reference control [The resulting cDNA was used to perform qPCR with PowerUp SYBR Green PCR Master Mix  and specific primers, reported in ry 2022) , and Pri control  and eachGAPDH) to normalize the values expressed as 2−ΔCT. Furthermore, we compared treated to untreated samples with the 2−ΔΔCT formula.Quantification of the levels of circRNAs and linRNAs was obtained using Bio-Rad CFX Manager Software and the cycle threshold (Ct) methods, determining the average of the PCR Ct duplicates. ΔCT method was used to compare each condition with the housekeeping gene (t-test (two-tail paired). The p-values and adjusted p-values  < 0.05 were considered statistically significant.The results of PCRs were analyzed with Student’s The circRNAs are molecules widely studied for their still-debated cellular roles. In this study, we investigated a host of circRNAs associated with cancer. Using two cell lines, we tested various anticancer agents to define a panel of drug-modulated circRNAs/linRNAs. In general, we observed a consistent upregulation of the circRNA and a concurrent downregulation of the linRNA that may be associable with different cellular responses, drug resistance, and cell survival. Interestingly, in MCF-7 cells an exclusive increase of circ/linVRK1 mediated by XL765 indicated effective treatment without upregulation of dangerous circRNAs, such as circ/linMAN1A2, which leads to greater motility, while in MDA-MB-231 cells AMG511 and GSK1070916 1 appeared to be more specific and able to decrease circGFRA1, as a good response to drugs.


High-content microscopyoffers a scalable approach to screen againstmultiple targets in a single pass. Prior work has focused on methodsto select “optimal” cellular readouts in microscopyscreens. However, methods to select optimal cell line models havegarnered much less attention. Here, we provide a roadmap for how toselect the cell line or lines that are best suited to identify bioactivecompounds and their mechanism of action (MOA). We test our approachon compounds targeting cancer-relevant pathways, ranking cell linesin two tasks: detecting compound activity (“phenoactivity”)and grouping compounds with similar MOA by similar phenotype (“phenosimilarity”).Evaluating six cell lines across 3214 well-annotated compounds, weshow that optimal cell line selection depends on both the task ofinterest and distribution of MOAs within the compound library. Given a taskof interest and a set of compounds, we provide a systematic frameworkfor choosing optimal cell line(s). Our framework can be used to reducethe number of cell lines required to identify hits within a compoundlibrary and help accelerate the pace of early drug discovery. High-content microscopy is a scalableapproach for characterizing the effect of small molecules on cells.5 In this setting, cellular responses to compounds are representedas feature vectors, whose entries measure observable changes, suchas in cell morphology, biomarker intensity, and localization. A single-passphenotypic profiling screen can provide annotation to compound librariesby identifying subsets of library compounds that show “phenoactivity”and by inferring MOA through “phenosimilarity” .9Large,diverse libraries of novel small molecules serve as criticalcomponents in the early drug discovery screening pipeline.10 aswell as general11 combinations of cellularreadouts for a given cell line. However, approaches for selectingoptimal cell lines are less explored.14 It is reasonable toexpect that different cell lines may have different sensitivitiesto detect different MOAs. For instance, others have focused on identifyingcompounds that induce differential responses across multiple celllines.16 However, it is poorly understood how toselect the best performing cell line for annotating diverse compoundlibraries in an unbiased- or target-agnostic fashion, and to whatextent using multiple cell lines would improve coverage.Phenotypic profiling screens depend critically on the selectionof cellular readouts and screened cell lines. For the purposes ofannotating large compound libraries, previous work has focused onselection of “optimal”Toexplore these questions, we first generated a high-content microscopydata set of six cell lines responding to a diverse set of 3214 smallmolecules with annotated MOA. We developed computational methods torank cell lines and combinations of cell lines according to theirability to infer compound activity and MOA. Lastly, we applied a classicoptimization framework to assess which cell lines or cell line combinationswould be best for annotating uncharacterized compounds.Our data set was generated usingsix cell lines. These included five cancer cell lines  from the NCI60 set of tumor cell lines; thesecell lines span a range of tissue types , monoblast and erythroid origins), cellularmorphologies, and are amenable to imaging assays. We additionallyincluded a noncancer patient-derived fibroblast cell line (“FB”).Additional details in Drug Repurposing Hub database and serve as an independent validation of our phenotypic profilingmethods. Compounds cover 664 MOAs (10We collected a “reference” library of 3214compounds annotated with mechanism of action (MOA). This library includesFDA-approved drugs, in-clinical-trial drugs, and established bioactivetool compounds. MOA annotations were curated from the 664 MOAs  Table S111 and sampled nine fieldsof view at 20×magnification for each well by microscopy. For each treatment, weperformed cell segmentation and feature extraction to generate 77quantitative features  phenosimilarity: thedegree to which the compounds with the same MOA share similar phenotypicprofiles  profiles .Our approach focused on three key challenges. First, quantificationof phenoactivity and phenosimilarity depend on analytical parameters,which can be challenging to tune across multiple cell lines. We aimedto limit the number of such parameters and ensure that results wererobust to these parameter choices. Second, cellular phenotypic changesfrom DMSO control can range from subtle to severe across differentcell lines. We aimed to ensure that our measurements of phenoactivityand phenosimilarity were sensitive across this spectrum and couldreport on responses that were similar to, but distinct from control.Third, compounds annotated to share a common MOA may induce phenotypicallydissimilar responses . We aimed to define “phenotypictightness” of an MOA in a manner that was robust to some degreeof compound heterogeneity.Based on these requirements, we choseto define phenoactivity andphenosimilarity at the MOA level rather than at a compound-by-compoundlevel. In brief, for each cell and MOA, we computed: (1) a phenoactivityscore by comparing the distributions of distances of the MOA and DMSOpoint clouds to the centroid of the DMSO point cloud; and (2) a phenosimilarityscore by comparing the tightness of the MOA point cloud relative tothe nearest neighbor point clouds of each MOA compound.17 In some cases, point clouds for collections of compounds annotatedwith the same MOA and DMSO replicates were similarly separated acrosscell lines, while in others dramatic cell line differences were apparent.For example, in the case of the HDAC inhibitors, both HEPG2 and OVCAR4cell lines showed similar degrees of separation between HDACs andDMSO, with both cell lines detecting phenoactivity for a similar subsetof HDAC compounds  that would be difficult to distinguishalterations for compact cells. In addition, overall morphology (geometry)features of the cell are an important driver of phenotypic variation,and HEPG2 is less variable in its geometry due to its colony growthpattern. This example of a poorly performing cell line highlightsthe importance of considering cell morphology when selecting a cellline of interest for phenotypic screening—and exemplifies theuse of our framework to select-out cell lines with these properties.How much improvement inphenoactivity detection is provided by inclusion of additional celllines? We compared the abilities of individual and pairs of cell linesto detect phenoactivity across all MOA categories 1d. For aWe next assessedwhether compoundswith the same provided MOA induce similar cellular phenotypes, whichwe quantified through phenosimilarity scores . As prevMOAs with high phenosimilarity in a given cellline weretightlyclustered and fell further from the DMSO point cloud 2a. MOAs 20 Thus, GSK3inhibition is expected to produce phenotypes in A549. Indeed, 8/11GSK3 inhibitors showed phenoactivity in A549, and clustered closelytogether in phenotypic space . In contrast to A549, increased GSK3 kinase activityhas not been reported in the noncancer FB cell line and GSK3 inhibitorsshowed low rates of phenoactivity and phenosimilarity.Differential phenosimilarityof GSK3 inhibitors between A549 andFB can be explained by the distinct roles of GSK3 in these 2 celllines. Nonsmall cell lung cancer cell lines, such as A549, exhibitincreased GSK3 kinase activity, which supports tumor cell proliferationand contributes to the poor prognosis.Moderatephenosimilarity scores for mTOR inhibitors  highlight the fact that while these compounds cluster nearone another in phenotypic space, they closely neighbor other compoundswith different MOA annotations. In the case of A549, a subset of closelygrouped mTOR compounds is reflected in the bimodality of the distancedistribution 2a-b. TheTo inspect whether cellularphenotypes induced by different MOAs were observable in images, weconsidered three well-studied categories: TP inhibitors, proteasomeinhibitors, and PLK inhibitors. As caveats: we note that MOA classesare better separated by considering the entire phenotypic profile,rather than changes in any single feature; a single cellular changemay be read out in multiple features ; and somefeatures may capture multiple effects observed in images . Nevertheless, these case studies provide an opportunityto examine whether cellular phenotypes corroborate prior work.23 Specifically,upon treatment we observe fragmented nuclei and increases in cellularsize in both cell lines . This is a well-described phenotype resultingfrom microtubule inhibition in the literature.ll lines b. Thesell lines a, iii-iv24 Treatment with PLK inhibitorswill often arrest cells in the G2/M phase, causing aberrant mitoticand postmitotic phenotypes and ultimately toxicity to fast-cyclingcancer cell lines.25 Consistent with thesefindings, PLK inhibitors show strong phenotypes associated with cellcycle defects in both our A549 and FB, with cells that have progressedpast G2/M displaying fragmented multiple small nuclei due to DNA segregationerrors .In our case study,we found that the best performing cell lineswere different for either identifying bioactive compounds or categorizingcompounds by MOA classes, and that the utility of using additionalcell lines was not the same for each of these tasks. A cell line thatis optimal to detect phenoactivity may have poor sensitivity for thesame MOA class (and vice versa). This is because the ability to inferMOA from clusters in phenotypic space requires that compounds withinthe target MOA are near one another yet far from compounds with otherMOAs. Phenosimilarity scores capture these two properties. In practice,large compound libraries could be annotated by evaluating how phenotypicallysimilar unknown compounds are to known reference classes. Phenosimilarityscores assess the degree to which reference classes are phenotypicallyconsistent and thus provide a measure of confidence for the degreeto which new, phenotypically similar compounds belong to a given MOA.10 In both cases we assumed that a reference compoundlibrary was provided with MOA annotation classes. A future task isto investigate how phenosimilarity scores can be used to refine orcoarsen provided MOA annotation classes. For instance, by replacingthe DMSO reference distribution with some other MOA class in our phenoactivityanalysis, one could evaluate the similarity between two MOA classesand provide guidance for when two MOA classes should be merged. Suchanalyses offer a new path to annotating compound libraries based onphenotypic consistency. A larger task, for future work, is an experimentaland computational platform designed to optimize over all three inputchoices for a given screening goal.There are a number of ways in which our approach could be improvedor extended. From a platform perspective, we made a number of technicalchoices, including compound dose, treatment time, cell features, constructionof phenotypic profile, distance metric, optimization framework, andso on. Each of these choices can be further examined for improvedperformance. From a more general perspective of using high-contentimaging to detect and predict MOA of uncharacterized compounds, threeare three major inputs to this process: cell line, biomarker, andannotated reference compound sets. Here, we optimized over cell lineswhile holding the biomarkers (cell painting) fixed, while in pastwork, we optimized over biomarkers while holding a cell line fixed.High-content microscopyscreens offer the promise to annotate largelibraries of uncharacterized compounds. However, the ability of differentcell models to classify compounds varies according to their underlyingbiology. Here, we show an objective framework for selecting one, ora small number of cell lines that are “optimal” forpredicting bioactivities across specified MOA classes. It is our hopethat this framework will help increase the scale, sensitivity, andaccuracy of phenotypic profiling used in early drug discovery.


Among the most malignant cancers, oral squamous cell carcinoma stands out as the most common malignant head and neck tumor, with high prevalence around the world, mainly in developing countries. Despite advances in the field of treatment, the prognosis of patients with oral squamous cell carcinoma remains poor, with a 5-year survival rate of less than 50%. Aiming to overcome the limitations of currently existing therapies, the present work proposes to investigate the potential of photodynamic therapy with methylene blue or toluidine blue. We found higher incorporation rates of both drugs in tumor cells compared to the non-tumor cell line. In addition, photodynamic therapy increases cytotoxic activity of both compounds for all lineages evaluated. Moreover, combination of both compounds promotes a synergistic effect on the viability of oral squamous cell carcinoma, opening up possibilities for the use of photodynamic therapy against other cancer cell types using combinations of these photosensitizers.50 value of 362.6 µM and 41.4 µM for MB and TB, respectively. Using PDT, all lineages showed greater sensitivity, presenting lower IC50 values when compared to the dark phase values. The combination index values of 0.69 (dark phase) and 0.73 (clear phase) associated with concave isobolograms, in both phases, revealed that MB and TB have synergistic effects when combined against SCC-9 cells. These findings suggest that MB or TB assisted with PDT holds promise for OSCC treatment.Among the most malignant cancers, oral squamous cell carcinoma (OSCC) stands out as the most common malignant head and neck tumor. Despite advances in the field of treatment, the prognosis of patients with OSCC remains poor. Aiming to overcome the limitations of the currently existing therapies against OSCC, the present work aims to investigate the potential of photodynamic therapy (PDT) with phenothiazine derivatives used alone or in combination. The incorporation of methylene blue (MB) and toluidine blue (TB) was evaluated in OSCC cell lines (HSC-3 and SCC-9) and a nontumor cell line (Hfib). Both compounds exhibited concentration and time-dependent incorporation, with higher rates observed in tumor cells. Regarding dark-phase cytotoxic activity, SCC-9 cells were the most sensitive cell line with an IC Among the most malignant cancers, oral squamous cell carcinoma (OSCC) stands out as the most common malignant head and neck tumor, with high prevalence around the world, mainly in developing countries ,2. OSCC An attractive option for the treatment of OSCC is the use of photodynamic therapy (PDT). PDT involves the use of a photosensitizer along with a special type of light that, when combined, induce the production of a form of oxygen that is harmful to various cell types, including tumor cells ,6,7. PDTAmong the various classes of photosensitizers, phenothiazines stand out for being molecules with great potential and propensity for the formation of reactive oxygen species . So far,Methylene blue (MB) and toluidine blue (TB) were purchased from Sigma-Aldrich . Both compounds were diluted in PBS and filtered with a 0.22 µm syringe filter. Both compounds were stored in a −20 °C freezer until use.2. All cell lines were regularly tested for mycoplasma contamination using MycoAlert® Mycoplasma Detection Kit .HSC-3 (human tongue squamous cell carcinoma) and SCC-9 (human tongue squamous cell carcinoma) cell lines were obtained from the American Type Culture Collection . Both cell lines were cultured in DMEM/F12 medium  supplemented with 10% fetal bovine serum , 400 ng/mL hydrocortisone (Sigma-Aldrich) and 50 µg/mL of gentamicin . The normal human epithelial fibroblast cell line (Hfib) was isolated from a healthy donor skin biopsy and cultured in DMEM medium (GIBCO) supplemented with 10% FBS and 50 µg/mL of gentamicin, as previously described . The cel4 cells/well in their respective medium described above for 24 h at 37 °C and 5% CO2. After that time, different concentrations of MB (12.5–400 µM) or TB (6.25–200 µM) were added to the plate, in triplicate, and the plates were incubated for different times . Then, the plates were washed twice with PBS and the cells were incubated with sodium dodecyl sulfate  to promote cell membrane lysis and release of MB or TB incorporated by the cells for 30 min in room temperature. The absorbance of the incorporated MB or TB was measured in a spectrophotometer at a wavelength of 660 nm. Three independent experiments were performed.Initially, all the cell lines were plated into 96-well plates at a cell density of 2 × 104 cells/well in in their respective medium described above for 24 h at 37 °C and 5% CO2. After that time, different concentrations of MB (12.5–400 µM) or TB (6.25–200 µM) were added to the plate, in quadruplicate, and the plates were incubated were at 37 °C for 2 h. Then, the plates were washed twice with PBS, the medium was replaced, and the plates were incubated at 37 °C for an additional 24 h. Finally, cell viability was measured using CellTiter-Glo® Luminescent Cell viability assay, which indicates cell viability by measuring ATP, following the manufacturer’s instructions. Both compounds were tested in the absence of light (dark phase) or after photodynamic exposure (clear phase) with a diode laser which has a semiconductor active medium InGaAlP  (The cells were plated into 96-well plates at a cell density of 2 × 10 Brazil) . The phoFor in vitro drug combinations, each one of the drugs was tested in eight concentrations alone (12.5–400 µM) or combined in a ratio of 1:1 or 2:1 MB/TB (3.12–200 µM) against SCC-9 cells following the protocol described above. The combined therapy was performed during both the dark and clear phase. The analysis of the combined effects was performed by determining the combination index (CI), used as cutoff to determine synergism, by using the Chou–Talalay CI method , and thr50 values. One-way analysis of variance and Newman–Keuls multiple comparison tests were used to determine the statistical significance of the group comparisons in the in vitro assays. The results were considered statistically significant when p was <0.05. All analyses were performed using Graph Pad Prism version 8.0 .We used non-linear regression for calculating the ICp < 0.05) of MB incorporation by non-tumor cells were only observed at the highest concentration evaluated (400 µM), which is different to what was observed in tumor cells that had significant rates (p < 0.05) of MB incorporation at concentrations ranging from 100 to 400 µM  was determined in oral squamous cell carcinomas (HSC-3 and SCC-9) and in a non-tumor cell line (Hfib). As revealed in o 400 µM . Among to 400 µM .p < 0.05) than the non-tumor cell line, especially at 100 or 200 µM  to excite MB, we found an increase in cytotoxic effect, with IC50 values of 234.5, 307.4 and 294.4 µM against HSC-3, SCC-9 and Hfib cells, respectively, versus IC50 values of >400, 362.6 and >400 in the dark phase, respectively (p < 0.05). MB-PDT is well employed against several types of cancer such as: cervical carcinoma, leukemia, osteosarcoma, mammary adenocarcinoma, melanoma and lung adenocarcinoma [Regarding cytotoxic activity in the dark phase, MB showed a significant inhibitory effect on cell viability, especially at 400 µM for all lineages, with the SCC-9 cell line being the most sensitive with an IC362.6 µM ; Table 1ectively . The difarcinoma ,36. Howearcinoma ,38,39. Parcinoma  demonstrIn comparison to MB, TB showed a more prominent cytotoxic effect on cell viability in the dark phase for all lineages, especially in concentrations between 50 and 200 µM . As reve50 values when compared to the dark phase values, again with the SCC-9 cell line showing the highest sensitivity with an IC50 value of 33.8 µM (p < 0.05). Similar results using TB-PDT were found using breast cancer cell lines, where TB presented an IC50 value of 1.13 µM in the dark phase and 0.88 µM in the presence of light, reinforcing the advantages of PDT [In the clear phase, all lineages showed greater sensitivity, presenting lower IC 33.8 µM . The lar 33.8 µM . The difs of PDT . Despites of PDT . It is as of PDT ,43.50 values of 368.5 and 43.8 µM when tested individually, and 30.5 and 26.9 µM when tested in combination, respectively. These data demonstrate an approximately 13-fold reduction in the IC50 value for MB and an approximately 2-fold reduction in the IC50 value for TB  and 0.73 (clear phase) associated with concave isobolograms, in both phases, revealed that MB and TB have synergistic effects when combined against the cell viability of SCC-9 cells . Interes of 0.89 . In addied alone . For treed alone ,47.In sum, MB or TB assisted with PDT as a useful strategy for the treatment of oral carcinoma was reinforced in the present investigation. In addition, it was shown that the combination of MB and TB has a synergistic effect on the viability of oral squamous cell carcinoma, opening up possibilities for the use of PDT against other cancer cell types using combinations of these phenothiazine derivatives already well characterized as promising photosensitizers.


From among the synthesized title compounds, 9,9-dimethyl-8-phenyl-9H-benzo [b]pyrrolo triazolodiazepine (7) was found most to be the most active compound exhibiting IC50 value of 6.60, 5.45, 7.85, 11.21, 12.24, 10.12, and 11.32 µM against MCF-7, MDA-MB-231, HeLa, SKOV-3, A549, HCT-116 and DLD-1 cell lines, respectively. Further the compounds were found to be non-toxic against normal human embryonic kidney (HEK-293) cell line.Herein, we report an efficient and eco-friendly, ultrasound assisted synthetic strategy for the construction of diversified pyrrolobenzodiazepine-triazole hybrids, which are potentially pharmaceutically important scaffolds, via a domino reaction involving intermolecular electrophilic substitution followed by intramolecular Huisgen 1,3-dipolar azide-alkyne cycloaddition. The USP of the reported protocol is the use of benign and inexpensive, recyclable molecular iodine-ionic liquid synergistic catalytic system cum reaction media for achieving the synthesis. The other salient features of this method are the use of mild reaction conditions, high yield and atom economy, operational simplicity, broad substrate scope and easy workup and purification. All the synthesized compounds were evaluated for  The classical methods for the construction of diverse fused heterocyclic compounds entail the sequential formation of the individual bonds in the target molecule. However, a more effective strategy is to form the various bonds in a single operation via domino/cascade or multi-component reactions (MCR), without separating the intermediates, altering the reaction conditions, leading to faster synthesis and low waste production, thus drastically reducing the amount of solvent, reagents, energy and labor used, as well as simplifying the purification process . Due to 4 is an inexpensive and commercially available, efficient, less toxic and recyclable ILs that has been successfully used in a variety of organic transformations. It has been reported to be superior to other commonly used imidazolium based ILs   Hussain. HoweverH-pyrroles (1a and 1b) were obtained from 2-azidoanilines through Paal-Knorr pyrrole synthesis while propargyl alcohols (2–5) were synthesized from aldehydes and terminal alkynes (H-pyrrole 1a and 4-(4-methoxyphenyl)-2-methylbut-3-yn-2-ol 2a under the influence of PTSA, oxalic acid, acetic acid and triflic acid (TfOH) at temperatures ranging from RT to 80°C. However, no reaction was observed with any of these catalysts   and faster reaction time was observed (When this reaction was carried at room temperature (RT) in [bmim]OH, [Bmim]Cl, [Bmim]Br, [Bmim]ClO4 and [Bmim]OAc on the yield and rate of the reaction. However, none of the other ionic liquids screened were found to be as effective as [Bmim]BF4. The effectiveness of these ionic liquids for the above reaction was observed to be in the order [bmim][BF4] > [Bmim]PF6 > [Bmim]Br >[Bmim]Cl > [Bmim]ClO4 > [Bmim]OAc > [Bmim]OH . Use of propargyl alcohols with electron releasing groups at para position of the phenyl ring attached to alkynyl carbon gave better yields  as compared to propargyl alcohols having electron withdrawing groups (9–10). Presence of electron releasing group at para position of phenyl ring attached to C-1 carbon of propargyl alcohols also greatly assisted in the product formation leading to enhanced yield of the products (Compounds 14–16). Furan ring at 1-position of propargyl alcohols also enhanced the yield of the product (Compounds 21–22). Presence of methyl substituents at 2 and 3 position of 1-(2-azidoaryl)-1H-pyrroles led to slightly lower yields  -1H-pyrroles (1a and 1b) anilines and a variety of substituted propargyl alcohols (2–5) were used as substrates to obtain the corresponding pyrrolobenzodiazepine-fused triazole hybrids. The corresponding 9 and 13) .The reaction is assumed to initiate through the activation of alcohol by iodine and Bmim+ by forming an interaction with the oxygen atom of the propargyl alcohol A. In the presence of strong π-nucleophile pyrrole B, an electrophilic substitution reaction then takes place at the C-2 position of the pyrrole ring (the most nucleophilic center on the pyrrole nucleus) to form an intermediate species C, which undergoes re-aromatization to form C-2 propargylated pyrrole D. Ultrasonic irradiation is thought to assist the intramolecular azide−alkyne 1,3-dipolar cycloaddition reaction in intermediate D furnishing the target molecule E .4 for the formation of 8-(4-methoxyphenyl)-9,9-dimethyl-9H-benzo [b]pyrrolo triazolodiazepine 6 using the reaction of 1-(2-azidophenyl)-1H-pyrrole 1a with 4-(4-methoxyphenyl)-2-methylbut-3-yn-2-ol 2a. After completion of the reaction, the reaction mixture was extracted thrice with ethyl acetate. The ionic liquid residue was washed with hexane and dried in vacuum to obtain pure [bmim][BF4] which was used for the next cycle. The recycled Bmim][BF4] so obtained could be used for the reaction up to four cycles with only a small drop in yield . Compound 7 also exerted significant anti-proliferative effect against MCF-7 and Hela cell line with IC50 values of 6.60 and 7.85 µM respectively. Compound 7 also exhibited good anti-cancer activity against SKOV-3, A549, HCT-116 and DLD-1 cell lines with IC50 of 11.21, 12.24, 10.12 and 11.32 µM respectively. Compound 6 exhibited moderate anti-proliferative against MCF-7, MDA-MB-231 and HeLa cell lines with IC50 of 12.58, 15.72, and 18.45 µM, respectively, and was found to be inactive against SKOV-3, A549, HCT-116 and DLD-1 cell lines. Compound 8 showed activity against SKOV-3 cell line (IC50 = 12.86 µM). Compound 8 also exhibited mild activity against MCF-7 (IC50 = 19.28 µM), MDA-MB-231 (IC50 = 14.48 µM), HeLa (IC50 = 18.30 µM), A549 (IC50 = 15.86 µM) and HCT-116 (IC50 = 16.72 µM) cell lines. Compound 12 was found inactive against DLD-1 cell line. Compounds 9, 10, 12, 15, and 16 showed good to mild activity against MCF-7, MDA-MB-231, HeLa and SKOV-3 cell lines in an IC50 range of 12.80–19.10 µM. These compounds were found inactive against A549, HCT-116 and DLD-1 cell lines. Compounds 13 and 14 exhibited mild activity against MCF-7 and SKOV-3 cell lines. Compound 14 exhibited good activity against HCT-116 and DLD-1 cell lines with the IC50 value of 12.68 and 10.82 µM. Compound 14 also showed moderate inhibition against HeLa cell line (IC50 = 15.10 µM) and was found inactive against MDA-MB-231 and A549 cell lines. Compound 11 exhibited good activity against MDA-MB-231(IC50 = 12.48 µM), mild activity against HeLa (IC50 = 12.48 µM) and weak activity against A549 (IC50 = 18.44 µM) cell lines. Compounds 17 and 18 were found inactive against HeLa and SKOV-3 cell lines while in other cell lines these compounds exhibited mild to weak inhibition. From the above discussion it is clear that compound 7 having un-substituted phenyl ring attached to the triazole moiety exhibited the highest anti-cancer activity. Introduction of methyl substituents on the fused benzene ring greatly reduced the anti-cancer activity , MDA-MB-231 (ER -ve breast cancer), HeLa , SKOV-3 (ovarian cancer), A549 (lung cancer), HCT-116 and DLD-1 (colon cancer) in order to test their anti-proliferative potential . Toxicit2S2O3 (1 M), washed with saturated solution of NaHCO3 and dried in vacuo to afford the crude product mixture, which was purified through column chromatography (EtOAc/hexane). The ionic liquid residue was washed with hexane and dried in vacuum to recover the [bmim][BF4] which was used in the next cycle.To a mixture of 1-(2-azidoaryl)-1H-pyrroles 1 (2 mmol) and propargyl alcohol 2–5 (2.5 mmol) and in [bmim][BF4] (4 ml) in a round bottom flask was added a catalytic amount of iodine (10 mol%) at room temperature, and the reaction temperature was allowed to rise to 40°C. The reaction was stirred at this temperature for the next 1 h and then it was subjected to ultrasound irradiation till completion of the reaction. The reaction mixture was now quenched with addition of water and extracted thrice with 10 ml of ethyl acetate. The combined organic extracts were treated with an aqueous solution of Na2) incubator. The compounds were then diluted to the desired concentrations in the culture medium. After 48 h, the media was removed and 100 µl MTT (0.5 mg/ml) solution was added to each well, and the plates were further incubated for another 3 h. Supernatant from each well was removed, formazan crystals were dissolved in 100 µl of dimethyl sulfoxide (DMSO) and absorbance was recorded at 540 nm wavelength using a spectrophotometer.The anti-cancer activity of the synthesized pyrrolobenzodiazepine-triazoles hybrids against various cancer cell lines was evaluated using MTT -2,5-diphenyl tetrazolium bromide) reduction assay . 2.5 × 14] synergistic catalytic system cum reaction medium under ultrasound irradiation. The disclosed protocol proceeds at ambient temperature, under ligand-, metal-, and base-free conditions, is operationally simple, atom economic and the ionic liquid reaction media can be recycled, further enhancing its green credentials. The synthesized molecules exhibited good in vitro anti-proliferative activity against different cell lines cancer cell lines in an IC50 range of 5.45–19.10 µM. Further, the evaluated molecules were found to be non-toxic against normal human embryonic kidney (HEK-293) cell line. Compound 7 exhibited the best anti-cancer activity with IC50 values of 6.60, 5.45, 7.85, 11.21, 12.24, 10.12, and 11.32 µM against MCF-7, MDA-MB-231, HeLa, SKOV-3, A549, HCT-116 and DLD-1 cell lines, respectively, and can be considered as a new anti-cancer lead molecule.In conclusion, a green and efficient domino synthetic strategy for the C-2 selective propargylation of pyrroles followed by intramolecular azide-alkyne Huisgen 1,3-dipolar cycloaddition leading to the construction of diversified pyrrolobenzodiazepine-fused triazole hybrids in high yields is reported. To the best of our information this is the first eco-friendly method for the synthesis of these biologically relevant hybrid scaffolds, and only the second synthesis of these molecules overall. The driving force for the present method is a simple and inexpensive, green, iodine-[bmim][BF


Chronic active Epstein–Barr virus (EBV) infection (CAEBV) is characterized by chronic or recurrent infectious mononucleosis-like symptoms and is associated with EBV-associated T/natural killer (NK)-cell lymphoproliferative disorders, which frequently lead to the development of life-threatening complications, such as virus-associated hemophagocytic syndrome and EBV-positive apparent leukemia/lymphoma mainly in T- and NK-cell lineages. In order to clarify the EBV genes responsible for the diseases, we introduced the plasmid coding sequences of EBV-encoded small RNAs (EBERs) and/or latent membrane protein (LMP) 1 into human T-lymphocyte virus-I-negative human T-cell lines using a gene expression vector harboring EBV nuclear antigen 1, established the G418-resistant transformants of five T-cell lines, and quantitatively examined the expression of EBERs and LMP1 using real-time reverse transcriptase–polymerase chain reaction. The expression levels of EBERs in T-cell transformants with EBER DNA paralleled those in EBV-positive human T- and NK-cell lines, SNTK cells. The expression of LMP1 mRNA varied in SNTK cells and in human T-cell transformants, and the expression of LMP1 mRNA in T-cell lines expressing both EBERs and LMP1 was much lower than that in the same cell line expressing LMP1 mRNA alone. The currently employed gene expression system and currently obtained transformants may be useful for the analyses of the pathophysiology of CAEBV and EBV-positive T/NK-cell lymphoproliferative disorders. Epstein–Barr virus (EBV) is a ubiquitous herpesvirus in humans. Primary EBV infection is generally asymptomatic, but it sometimes causes infectious mononucleosis, which is basically self-limited. However, some individuals, mostly in East Asia, develop chronic infection with EBV. Chronic active EBV infection (CAEBV) is characterized by chronic or recurrent infectious mononucleosis-like symptoms and by high viral loads in peripheral blood ,2, and iIn CAEBV, the transcripts of EBV latent infection genes are detected in T or NK cells , and theBamHI-A rightward transcripts (BARTs), latent membrane protein (LMP) 1, and LMP2s are expressed in infected T and NK cells in CAEBV [Profiles of the expression of the EBV latent infection genes in EBV-infected T and NK cells exhibit variations, and EBV nuclear antigen 1 (EBNA1), EBERs, in CAEBV ,13. EBNAin CAEBV ; furtherin CAEBV . In the current study, we aimed to establish cell models in which we could evaluate the role of EBV latent infection genes under a condition where the effects of other factors were minimized, which might affect host cellular gene expression. We thus introduced the plasmid coding sequences of EBERs and/or LMP1 into HTLV-I-negative human T-cell lines using a gene expression vector harboring EBNA1, and we established stable transformants.TM PLUS  with EBV from B95-8 cells . HTLV-I-negative human T-cell lines, MOLT-14 and Peer, were gifts from Fujisaki Cell Center, Hayashibara Biochemical Labs., Inc. . CCRF-HSB2, also an HTLV-I-negative human T-cell line, was a gift from the Health Science Research Resources Bank . Human T-cell lines, Jurkat [A lymphoblastoid cell line (LCL), TK6, was established via the infection of peripheral blood lymphocytes prepared from a healthy donor using Ficall-Paque, Jurkat  and MOLT, Jurkat  . The 0.75 kb NheI-SacI fragment of pEGFP-C1 containing the green fluorescence protein (GFP) open reading frame was cut out, and at the 5′-end of the fragment, an XhoI site was added; subsequently, the fragment containing the GFP open reading frame was subcloned into the XhoI site in multiple cloning sites (MCSs) of pEBMulti-Neo (pEBMulti-Neo-GFP). The 1.0 kb SacI-EcoRI subfragment of the EcoRI K fragment (EKS) of Akata EBV DNA, which corresponds to the EcoRI J fragment of B95-8 EBV DNA and contains EBER1 and EBER2 reading frames [EcoRI K fragment (EK) of Akata EBV DNA. At each end of the EKS fragment, a BglII or BamHI site was added, and the BglII-BamHI fragment containing the EKS fragment was concatenated six times in a direction-restricted fashion at the BamHI site in the MCSs of pEBMulti-Neo (pEBMulti-Neo-EKS6) [BamHI fragment of pSV2gptMTLM  containing EBV LMP1 cDNA [BamHI site in the MCSs of pEBMulti-Neo (pEBMulti-Neo-LMP1).A gene expression vector, pEBMulti-Neo, harboring g frames , was cuteo-EKS6) . The 3.0MP1 cDNA  was cut 6 cells were suspended in serum-free RPMI 1640 medium, washed twice, and resuspended in 600 μL of ice-cold medium containing 20 μg of plasmid DNA in a 4 mm gap electroporation cuvette. Electroporation was performed with Gene Pulser II  at 1 mF and at the optimal voltage (210 V to 270 V) for each cell line [4 cells were seeded in a well in flat-bottomed 96-well culture plates with 200 μL of selection medium. Half of the medium was changed every 5 days until colonies emerged. Clones were expanded and maintained in the selection medium.For transfection, plasmid DNA was purified with a FlexiPrep kit  or an illustra plasmidPrep Midi Flow kit . Plasmids were introduced to cell lines via electroporation. Then, 5 × 10ell line . The vol260, and subsequently treated with DNase using a TURBO DNA-freeTM kit . For the reverse transcription of RNA samples, five μg of DNase-treated total RNA was converted to cDNA with a High-Capacity cDNA Reverse Transcription kit  in a 50 μL reaction volume containing random hexamers according to the manufacturer’s protocol. For real-time PCR, a 0.5 μL aliquot of the cDNA sample  was diluted into 20 μL of a solution containing TaqManTM Fast Advanced master mix  and each TaqMan assay reagent described below. Real-time PCR amplification was performed using the QuantStudio 3 real-time PCR system , according to the manufacturer’s protocol. Total RNA was extracted from the cell lines using TRIzol reagent , quantified by measuring ODFor the absolute quantification of the copy numbers of EBERs, primers and TaqMan MGB probes were designed with Primer Express  . To checFor a relative quantification of LMP1 mRNA, the primers and TaqMan MGB probe were synthesized as previously described  Table 1Table 1. We could establish the G418-resistant transfectants of all five T-cell lines with the transfection of pEBMulti-Neo-GEP , pEBMulti-Neo-EKS6 , pEBMulti-Neo-LMP1 , or both pEBMulti-Neo-EKS6 and pEBMulti-Neo-LMP1 . In the case of the transfection of both pEBMulti-Neo-EKS6 and pEBMulti-Neo-LMP1, 10 μg of each plasmid, totaling 20 μg, was used. For the analysis of RNA expression in transformants, cells were cultured for one week without G418; then, the cells were harvested, and the cell pellets were frozen quickly in liquid nitrogen and stored at −80 °C until use.The expression levels of EBERs in SNTK cells showed variations, but, generally, they were equivalent to or much higher than those in LCLs. Furthermore, the copy number of EBER1 was much higher than that of EBER2 in all SNTK cell lines and LCLs . In all Next, we examined the expression of EBERs in human T-cell lines with the co-expression of LMP1. The human T-cell transformants transfected with both pEBMulti-Neo-EKS6 and pEBMulti-Neo-LMP1 expressed LMP1 mRNA, even at a lower level than those with pEBMulti-Neo-LMP1 alone . The expThe expression of LMP1 mRNA varied in SNTK cells and in human T-cell transformants, and it was not detected in SNK1, SNK6, or SNK10 cells . The expIn the current study, we used a pEBMulti-Neo vector to introduce EBV latent infection genes, established stable transformants of HTLV-I-negative T-cell lines expressing EBERs and/or LMP1, and further examined the amount of expressed RNAs using real-time RT-PCR. pEBMulti-Neo is a gene expression vector harboring EBNA1 derived from EBV; thus, it is distributed to daughter cells via the episomally replicating system. Therefore, we did not have to consider the effects of plasmid integration sites in chromosomal DNA on the expression of host genes, and, furthermore, this expression system may mimic EBV infection itself.TM system , and we obtained the transformants of MOLT14 cells expressing EBERs, but the amount of expressed EBER1 was much lower than that in an EBV-positive Burkitt’s lymphoma cell line, Akata [Both EBER1 and EBER2 were expressed in all the cell lines carrying EBER genes, EBV-positive T- or NK-cell lines, and human T-cell lines transfected with pEBMulti-Neo-EKS6, and the amount of expressed EBER1 was much higher than that of EBER2 in all the cell lines examined, including in LCLs and EBV-producing B95-8 cells . The expe, Akata . Furthere, Akata . Furthere, Akata ; thus, tAmong the stable transformants established in this study, EBER1 expression generally increased with the expression of LMP1 mRNA, but EBER2 expression generally decreased with LMP1 mRNA . We did The expression of LMP1 mRNA in T-cell transformants introduced with both of the expression vectors for LMP1 and EBERs was much lower than that in the same mother cell line introduced with the LMP1 gene alone . In a B-The currently established transformants have some limitations as a cell model for evaluating the role of EBV genes, since we cannot control the expression level of the introduced genes. The expression level of EBV genes varied among T-cell lines, as well as among SNTK cells, especially in LMP1 mRNA . We are In the current study, we easily established stable transformants of human T-cell lines and observed the expression of EBV latent infection genes. The currently employed gene expression system and currently obtained transformants may be useful for the analyses of the pathophysiology of CAEBV and EBV-positive T/NK-cell lymphoproliferative disorders.


PIK3CA mutations, activating the phosphatidylinositol 3-kinase (PI3K) signaling pathway seem to be associated with chemotherapy resistance and poor outcome. Inhibition of the PI3K signaling pathway may lead to sensitization and prevention of the development of resistance to cytotoxic drugs. The present study aimed to investigate the anti-tumor activity of low-dose vinorelbine (VRL) combined with alpelisib, an α-selective PI3K inhibitor and degrader, in breast cancer (BC) cells. Human BC cell lines MCF-7, T-47D , MDA-MB-231 and BT-549  were exposed to a combination of low-dose VRL and alpelisib for 3 and 7 days. Cell viability was detected by the Alamar blue assay, and cell proliferation was determined by the BrdU incorporation. The effect of the substances on the p110α protein expression that is encoded by PIK3CA gene was investigated by Western blot. Low-dose VRL plus alpelisib showed synergistic anti-tumor effects and significantly inhibited cell viability and proliferation of MCF-7 and T-47D cells. Even lower alpelisib concentrations (10 ng/ml and 100 ng/ml) combined with low-dose metronomic VRL led to a significant reduction of cell viability of PIK3CA-mutated cells, and the anti-tumor activity was comparable with the effects at 1000 ng/ml alpelisib. Cell viability and proliferation of MDA-MB-231 and BT-549 cells were inhibited by VRL but not by alpelisib alone. This indicates that alpelisib did not significantly affect the cell growth of triple-negative, PIK3CA wild-type BC cells. The p110α expression was downregulated or not affected in PIK3CA-mutated cell lines, and not significantly upregulated in PIK3CA wild-type cell lines. In conclusion, combination of low-dose metronomic VRL and alpelisib showed synergistic anti-tumor effects and significantly inhibited the growth of HR-positive, HER2-negative, PIK3CA-mutated BC cells, providing a rationale for further efforts to evaluate this combination in vivo.In metastatic breast cancer (MBC),  With an incidence of 2.3 million and a mortality of 0.7 million cases per year, female breast cancer (BC) represents the most commonly diagnosed cancer and the fifth leading cause of cancer mortality worldwide . In thePIK3CA gene, which encodes the p110α subunit of PI3K, is the most frequently mutated gene -positive, human epidermal growth factor receptor 2 (HER2)-negative advanced BC /AKT/mammalian target of rapamycin (mTOR) signaling pathway plays one of the key roles in the regulation of cellular growth, metabolism, migration, and survival . PIK3CAal., 2020; Stemke-al., 2020; Xiao etal., 2020). The real., 2020; Mosele al., 2020; Rasti eal., 2020; Sobhanial., 2020; Yang etal., 2020). Conseqal., 2019). Metronal., 2019; Krajnakal., 2019; Montagnal., 2019). Based PIK3CA gene), T-47D , MDA-MB-231 and BT-549 , were all obtained from American Type Culture Collection . BC cells were cultured in RPMI 1640 culture medium , containing 10 % fetal calf serum and 1 % penicillin-streptomycin. Cells were incubated in a moistened atmosphere at 37 °C and 5 % CO2. For all functional assays, cells were detached by trypsin-ethylenediaminetetra-acetic acid  and seeded in 96-well plates  at 3,000 cells/well (passage 10/semiconfluence). VRL , (stock solution 10 mg/ml) diluted in Aqua dest., was added at levels corresponding to the concentration in serum of metronomically treated patients (0.63-5 ng/ml VRL), i.e. at much lower concentrations compared to maximum tolerated dose of conventional chemotherapy . The treatment with both substances was performed continuously for 3 and 7 days to simulate the metronomic dosing schedule. Human BC cell lines MCF-7 . In add2 in aluminum foil due to the photosensitivity of the redox dye for fluorescence measurements. The absorbance was measured at 590 nm (650 nm reference wavelength) using a GloMax® multi detection system microplate reader .Cell viability was measured using the Alamar blue assay kit (Thermo Fisher Scientific). After 3 and 7 days of treatment, 100 µl/well Alamar blue Cell Viability Reagent solution [(1:10 in Dulbecco's Phosphate-Buffered Solution (DPBS)] was added to the cell cultures and incubated for 4 hours at 37 °C and 5 % CO2. Afterwards supernatants were discarded and 200 ml/well Fix-Denat solution and 100 μl/well anti-BrdU antibody solution (1:100 in antibody dilution solution) were added. After incubation for 60 min at room temperature the absorbance was measured at 450 nm (690 nm reference wavelength) using a GloMax® multi detection system microplate reader (Anthos Labtec Instruments).Cell proliferation was detected by the Bromdeoxyuridine (BrdU) incorporation kit . 10 μl/well BrdU labeling solution (1:100 in DPBS) was added to the cells and incubated for 3 hours at 37 °C and 5 % CO2 cell culture plates. The cell number was chosen according to the proliferation rate so that the culture dishes were semiconfluent at the time of protein extraction. One day after seeding, the cells were treated for 7 days. For protein extraction, cells were washed with DPBS and mechanically removed with a cell scraper. The solution was centrifuged at 1200 rpm for 5 minutes. The supernatant was discarded and the pellet was dissolved with up to 1 ml of lysis buffer. The solution was transferred into a 2 ml reaction tube and placed on ice. After incubation for 30 min on ice the samples were centrifuged for 10 min at 14000 g. The supernatant was transferred to a new tube and stored at -20 °C. The BCA Protein assay kit (Thermo Fisher Scientific) was used to determine the protein concentration of the extracts. Equal amounts of protein (12.5 μg per lane) were separated by size using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) with 10 % polyacrylamide gels. Gels were transferred on polyvinylidene difluoride (PVDF) membrane by semi-dry blotting. Membranes were blocked for 1 hour according to the antibody manufacturer's instructions and then incubated with a primary antibody in blocking solution overnight at 4 °C on a roll mixer. The monoclonal antibody against PI3K p110α  was used at a dilution of 1:500. β-actin antibody (Sigma Aldrich) was employed at a dilution of 1:2000. After washing, the membranes were incubated with HRP-linked secondary antibody  at a dilution of 1:4000 (PI3K p110α Western blot) or 1:2000 (β-actin Western blot) for 1 hour at room temperature, and after washing the bound antibodies were visualized by adding of an enhanced chemiluminescent solution  and detected in a chemiluminescence detector . For quantification bands were quantified by densitometry evaluation using a computer-based pixel counting system . These values were referenced to the corresponding β-actin values of the same membrane as a loading control.To prepare protein extracts from cell culture, tumor cells were seeded on 143 mm t-test  was used to evaluate the statistical significance of the results. All p-values represented two-sided tests and statistical significance was assumed at a value of p<0.05. To quantify the effects of combination treatment, the combination index (CI)-isobologram equation based on the Chou-Talalay method was used . The graphical presentation was performed as a classical isobologram with isoboles of the IC50 or IC80 and presentation of the corresponding CI values in relation to the corresponding isoboles within the diagram.All experiments were performed in triplicates and repeated three times. The absorbance in Alamar blue and BrdU assay for the untreated control group was regarded as 100 % cell viability and 100 % cell proliferation, respectively. The results of absorbance for the treated groups were indicated as the mean ± standard deviation of three separate experiments and displayed as percentages relative to that of the control group. Student'shou, 2006). Based 1 and (D)2: required concentrations of the active compounds in combination to achieve the defined effect.(Dx)1 and (Dx)2: required concentrations of each active compound considered individually to achieve defined effect. where CI = combination index (CI). (D)PIK3CA-mutated cell lines, the combination of low-dose VRL and alpelisib decreased cell viability with increasing concentrations after both 3 and 7 days of treatment  and in a concentration corresponding to a lower alpelisib concentration . In all cell lines tested DMSO at concentrations 0.1-0.5 % did not significantly affect the cell viability Figure 2. HoweverPIK3CA-mutated cell lines, the combination of low-dose VRL and alpelisib decreased cell proliferation with increasing concentrations after 7 days of treatment  after 7 days of treatment  and the IC80 value at 3.43 ng/ml VRL (CI=0.685)  and IC80 (CI=1.190) value  and the IC80 at 4.79 ng/ml VRL (CI=0.906) . Taselial., 2017). In parPIK3CA-mutated cells, and the anti-tumor activity was comparable with the effects at 1000 ng/ml alpelisib. This indicates that above a certain concentration of VRL and alpelisib, no significant potentiation of the anti-tumor effect could be observed. The combination of the agents in lower doses may reduce side effects, so the efficacy and tolerability will be further evaluated in vivo.Serum concentration achieved in patients taking 300 mg alpelisib once daily on a regular basis averages 1000 ng/ml . Here, PIK3CA-mutated MBC patients. However, the combination did not demonstrate clinical utility given its safety profile and modest clinical benefit , suggesting that the PIK3CA gene may represent a predictive biomarker for alpelisib activity in this population . Since endocrine therapy combined with CDK4/6i has become the standard first-line treatment for HR-positive, HER2-negative MBC, a phase II trial, BYLieve (NCT03056755), was conducted to evaluate alpelisib plus fulvestrant in MBC patients who had progressed on immediate prior CDK4/6i plus aromatase inhibitor. In a cohort A, alpelisib plus fulvestrant met its primary endpoint, with 50.4 % of patients alive without progression after 6 months of treatment  via loss of phosphatase and tensin homolog (PTEN), a direct counterpart of PI3K . Therefal., 2021). Combinal., 2021).PIK3CA-mutated MBC patients, there is a promising approach to investigate alpelisib with chemotherapeutic agents as well. VRL, an orally available anti-microtubule agent, represents a standard treatment option in MBC . In addal., 2019; Liu et al., 2019). Based al., 2019; Krajnakal., 2019). Alpelial., 2014). As thePIK3CA mutation and triple-negative cell lines without a PIK3CA mutation were analyzed, so we cannot clearly state whether the significant reduction in the cell viability and proliferation shown depended on the PIK3CA mutation alone regardless of HR and HER2 status. Although we hypothesize that the PIK3CA mutation was primarily responsible for the synergistic effects and the effect of alpelisib shown, we consider the evaluation of HR-positive, HER2-negative cell lines without PIK3CA mutation as well as the evaluation of triple-negative cell lines with a PIK3CA mutation to be of particular importance.In this study, HR-positive, HER2-negative cell lines with a PIK3CA-mutated BC cell lines. In addition, we showed that even lower doses of alpelisib in combination with low-dose VRL result in significant anti-tumor effects, providing a rationale for further efforts to evaluate this combination in vivo with the intent of improving the toxicity profile.In conclusion, the present work revealed a significant reduction of cell viability and proliferation with synergistic effects of low-dose metronomic VRL and alpelisib in HR-positive, HER2-negative, SK received speaker honoraria from Roche Pharma AG and Novartis Pharma GmbH Germany, research funding from Novartis Pharma GmbH Germany and travel reimbursement from PharmaMar and Novartis Pharma GmbH Germany.KA received speaker honoraria from Clovis Oncology, MSD und AstraZeneca.ASH received speaker honoraria from Pfizer Pharma GmbH and honoraria from Medupdate GmbH.MS received honoraria for speaker or consultancy role from AMGEN, AstraZeneca, Eisai, Lilly, Myelo Therapeutics, Novartis, Pantarhei Bioscience, Pfizer, and Roche. He received research funding from AstraZeneca, BioNTech, Eisai, Genentech, Myelo Therapeutics, Novartis, Pantarhei Bioscience, Pfizer, Pierre-Fabre, and Roche Pharma AG. He received travel reimbursement from Pfizer and Roche. In addition, MS has a patent for EP 2390370 B1 issued and a patent for EP 2951317 B1issued.MJB received honoraria and expenses from Astra Zeneca, Clovis Oncology, GSK, MSD, Pharma Mar, Roche Pharma AG and Tesaro Bio Germany GmbH. He is consultant to Eisai, GSK, MSD, Pharma Mar, Roche Pharma AG and Tesaro Bio Germany GmbH. He received funded research from AstraZeneca, Clovis Oncology, MSD and Novartis.AH received honoraria from AstraZeneca, Celegen, MedConcept Gm, Med update GmbH, Medicultus, Pfizer, Promedicis GmbH, Pierre Fabre, Softconsult, Roche Pharma AG, Streamedup!GmbH and Tesaro Bio Germany GmbH. She is a member of the advisory board of PharmaMar, Promedicis GmbH, Pierre Fabre Pharma GmbH, Roche Pharma AG and Tesaro Bio Germany GmbH. She received research funding from Celgene. All remaining authors declare that there are no conflicts of interest regarding the publication of this manuscript.Conceptualization: SK, MJB and WB; investigation and formal analysis: SK, JPT, PFH and WB; writing - original draft: SK and WB; writing - review and editing: SK, KA, ASH, AL, MS, AH and MJB.The presented results are part of the doctoral thesis of Mr. Jannis Patrik Trier and Mrs. Pauline Friederike Heinzmann.The research work was conducted as a part of “Excellent Researchers in Breast Cancer” research project and was supported by Novartis Pharma GmbH Germany.