Mammary tumors are common in cats, presenting an aggressive behavior with high tumor recurrence. Therefore, new and efficient therapeutic protocols are urgent. Monoclonal antibodies  are widely used in human breast cancer therapy, inhibiting the HER2 dimerization and leading to cell apoptosis. Furthermore, drug combinations, with tyrosine kinase inhibitors (TKi) are valuable in patients’ therapeutic protocols. In this study, two mAbs, and an ADC, as well as combined protocols between mAbs and mAbs plus lapatinib (TKi) were tested to address if the drugs could be used as new therapeutic options in feline mammary tumors. All the compounds and the combined treatments revealed valuable antiproliferative effects, and a conserved cell death mechanism, by apoptosis, in the feline cell lines, where the mutations found in the extracellular domain of the HER2 suggest no immunotherapy resistance.her2 ECD (subdomains II and IV) was analyzed in 40 clinical samples of FMC, in order to identify mutations, which can lead to antibody resistance or be used as prognostic biomarkers. Results obtained revealed a strong antiproliferative effect in all feline cell lines, and a synergistic response was observed when combined therapies were performed. Additionally, the mutations found were not described as inducing resistance to therapy in breast cancer patients. Altogether, our results suggested that anti-HER2 mAbs could become useful in the treatment of FMC, particularly, if combined with lapatinib, since drug-resistance seems to be rare.Feline mammary carcinoma (FMC) is a highly prevalent tumor, showing aggressive clinicopathological features, with HER2-positive being the most frequent subtype. While, in human breast cancer, the use of anti-HER2 monoclonal antibodies (mAbs) is common, acting by blocking the extracellular domain (ECD) of the HER2 protein and by inducing cell apoptosis, scarce information is available on use these immunoagents in FMC. Thus, the antiproliferative effects of two mAbs (trastuzumab and pertuzumab), of an antibody–drug conjugate compound (T-DM1) and of combined treatments with a tyrosine kinase inhibitor (lapatinib) were evaluated on three FMC cell lines . In parallel, the DNA sequence of the  Similarly to human breast cancer , the felher2 [The epidermal growth factor receptor 2 (HER2) is a common target in patients with HER2-overexpression tumors . HER2 isher2 ,13, the Nowadays, the use of mAbs are widely common in HER2-positive breast cancer patients, decreasing its downstream pathways activation, such as AKT and mTOR, responsible for the antiapoptotic mechanisms , cell cyIn human breast cancer, 50% of the HER2-positive patients show immunotherapy resistance , the comher2 ECD (subdomains II and IV); (3) describe the cell death mechanism induced by the mAbs and T-DM1, in the carcinoma cell lines; (4) evaluate the synergistic antiproliferative effects by the combination of mAbs (trastuzumab plus pertuzumab) and assess the increase in the cytotoxic response by the use of mAbs with the TKi (lapatinib); (5) identify genomic mutations in the feline her2 gene ECD (subdomains II and IV) in FMC clinical samples, in order to recognize possible therapy resistant animals, or prognostic factors.An early diagnosis and individual therapy is crucial for the improvement of survival time  and prevv/v) CO2 , in Dulbecco’s Modified Eagle Medium , for CAT-MT and SKBR-3, whereas FMCm and FMCp cell lines were maintained in Roswell Park Memorial Institute 1640 Medium , both supplemented with heat-inactivated 20% (v/v) fetal bovine serum . Periodically, all cell lines were inspected to control their morphology and proliferation rate, being tested for Mycoplasma .In this study, three feline cell lines  ,51 and a3 cells/well for CAT-MT and FMCp, 15 × 103 cells/well for FMCm and 10 × 103 cells/wells for SKBR-3), and then exposed to increasing concentrations of each antibody , using the Cell Proliferation Reagent WST-1  and following the manufacturer’s instructions. Briefly, cell lines were seeded in 96-well plates to obtain a confluency of 90%, after 24 h  and pertuzumab plus lapatinib, a similar methodology was used, testing concentrations that covers different cytotoxic responses .Expression analysis of HER2 was performed, as reported by us ,52, in tHER2 immunoreactivity was scored as recommended by the American Society of Clinical Oncology’s (ASCO) , and as 4 cells/well for CAT-MT and FMCp, 10 × 104 cells/well for FMCm, and 7 × 104 cells/well for SKBR-3 cell lines), and then were exposed to mAbs and to the ADC for 72 h, at a concentration close to the EC50 value. Control wells were left unexposed, with the PBS used as a vehicle, in three independent experiments. The percentage of apoptotic cells after drug exposure was calculated by using the APC Annexin V Apoptosis Detection Kit with Propidium Iodide  and following the manufacturer’s instructions. Briefly, supernatants were harvested, and the remaining attached cells were trypsinized  and added to the correspondent supernatants. Then, samples were centrifuged for 5 min at 500 g at RT, washed with PBS and resuspended in 500 µL of Annexin V Binding Buffer (BioLegend), with a maximum concentration of 1 × 107 cells/mL. Afterwards, 100 µL of each sample were passed to a new tube, and 5 µL of APC Annexin V (BioLegend) and 10 µL of PI (BioLegend) was added. Then, samples were vortexed and incubated for 15 min at RT, protected from the light. Finally, 400 µL of Annexin V Binding Buffer (BioLegend) were added to the samples, before acquisition in a BD LSR Fortessa X-20 , at Champalimaud Foundation, Lisbon, Portugal. Data were analyzed using FlowJo software , considering double negative staining cells, as living cells, annexin positive cells in early apoptosis and double positive cells (both for annexin and PI) in late apoptosis phase.The flow cytometry assay was performed as already reported by us . For thiher2 gene amplification was not observed [The 40 tumor tissue samples used in this study were collected from cats that underwent mastectomy, at the Teaching Hospital of the Faculty of Veterinary Medicine, University of Lisbon, with all the procedures consented by the owners, showing no interference in the animals’ well-being. The clinical history of the cats was recorded , includiher2 ECD (NC_018736.3), previously identified by comparison with the genomic human her2 sequence (NC_000017.11), different primers were designed (Genomic DNA extraction was performed in 5 mg of 44 frozen tissue samples (4 breed control and 40 tumor tissues) and in the three feline cell lines , collected after grown till confluency in a T25 culture flask, as previously described ,55, usindesigned  in the Pher2 (NC_018736.3), using the ClustalW tool (BioEdit Alignment Editor software) [her2 sequence (NC_000017.11) and searched in National Cancer Institute, International Cancer Genome Consortium and Catalogue of Somatic Mutations in Cancer (COSMIC) databases for putatively induced resistance to mAbs, and the ADC tested in this study.The feline sequenced samples were aligned with the identified feline oftware) , while toftware) . Proteinp-values below 0.05 being considered statistically significant, and a 95% confidence interval . For the cytotoxicity assays, outliers with more than two standard deviations were removed from the analysis and the EC50 value for each drug, in all the cell lines was calculated using Log (agonist) vs. Response (Variable slope) function. In the drug conjugation assays, the two-way ANOVA test was performed. Regarding the mutations found in tissue samples, associations between groups were assessed using the nonparametric Fisher’s exact test.Statistical analysis was carried out using the GraphPad Prism software , with two-tailed 50 = 3047.89 µg/mL ± 1.43; 50 = 528.45 µg/mL ± 1.14) and FMCp  cell lines. In parallel, pertuzumab showed similar results on CAT-MT and FMCm cell lines . Regarding the ADC T-DM1, the highest antiproliferative effect, at the range of concentrations tested, was reported on CAT-MT and FMCp cells  = 52.84 µg/mL ± 1.50).Trastuzumab, pertuzumab and T-DM1 exhibited a dose-dependent antiproliferative effect on feline mammary carcinoma cell lines. Indeed, trastuzumab exerted a more potent antiproliferative effect on CAT-MT cell line  or ADC (T-DM1) allowed to confirm that the observed antiproliferative effects were related to apoptosis induction . Deepenip < 0.001; p < 0.001; p = 0.018; p < 0.001; The combination trastuzumab plus pertuzumab showed synergistic antiproliferative effects in all feline cell lines. In CAT-MT cells the exposure to trastuzumab at 125 µg/mL plus pertuzumab at 2000 µg/mL increased the cytotoxicity from 13.6% to 40.0%  combined with lapatinib showed an improvement of the antiproliferative effects induced by mAbs. Accordingly, the combination of trastuzumab plus lapatinib, in the CAT-MT cell line, revealed a valuable synergistic response, for all the concentrations tested, with the highest increase of 78.4% of cytotoxicity, by the combination of trastuzumab at 125 µg/mL plus lapatinib at 7.26 µg/mL  presented at least one mutation  of the mutations were located in exon 3 (11/40 tumor samples), no mutations were found in the exon 4. Additionally, exon 3 presented 11 synonymous and 10 missense mutations, the remaining four being silent mutations. Regarding the subdomain IV, which is the her2 region recognized by trastuzumab, few mutations were detected, namely one heterozygous missense mutation at exon 10 , in two tumor samples; five mutations in exon 11 , one being a heterozygous synonymous mutation and four missense mutations, occurring in three tumor samples; two mutations at exon 12 , occurring in two tumor samples, one of them synonymous; three mutations at exon 13 , in two tumor samples, one synonymous, one missense and one frameshift mutation (c.14406 Ins. C).Previous studies on somatic mutations in the mutation , with alher2 ECD sequence, and searching in the referred databases, the mutations found were never reported as inducing resistance to anti-HER2 immunotherapy in breast cancer patients.Additionally, any correlation was reported between animals’ clinicopathological features and the described mutations. Moreover, after comparing the feline mutations with the genomic human The FMC is a common disease, showing similar features with human breast cancer ,2. Prese50 = 8756 nM ± 83), and a maximum of 79.4% of cytotoxicity in the FMCp cell line, by exposure to neratinib [Currently, only one felinized mAb is known to be in use as a therapeutic option for cats ,60, witheratinib , no clineratinib . Thus, teratinib , inducineratinib , and theeratinib , a crucieratinib ,68, beineratinib  decreaseeratinib . This reeratinib . Furthereratinib ,72. Perteratinib , with a eratinib  and ERK eratinib , which ieratinib . Finallyeratinib , which ieratinib ,40,77, beratinib . In facteratinib , suggesteratinib ,78,80, oeratinib ,82. Unfoeratinib ,83.The tested mAbs and the ADC cell death mechanism occur by apoptosis, suggesting a Bcl-2 dependent mechanism , conservAs for human breast cancer, combined protocols reveal to be a valuable tool ,86,87 inher2 ECD could explain the resistance patterns obtained with the use of anti-HER2 molecules, although mutations in the her2 gene are described as more common in the TK domain [her2 ECD mutations occurred in exon 3 (69.4%), subdomain II, which encode for the region recognized by pertuzumab, suggesting that if resistance occurs in cat, it might be more common in therapeutic protocols using this mAb. Additionally, 38.9% (14/36 mutations) of the mutations were synonymous and 61.1% (22/36 mutations) corresponds to missense mutations, conditioning 59.1% (13/22 mutations) of them a change in the polarity of the codified codon, which could modify the 3D arrangement of the protein [her2 ECD is S310F/Y, related to a resistant pattern to trastuzumab [her2 ECD were described as inducing resistance to therapy, according to National Cancer Institute, International Cancer Genome Consortium and COSMIC databases, and none of them were related to the cat’s clinicopathological features.Beyond the acquired therapy resistance, somatic mutations in the K domain , and in K domain . In the  protein . In breastuzumab  and pertstuzumab ,102 therThis study tested two mAbs (trastuzumab and pertuzumab) and an ADC compound (T-DM1) revealing valuable in vitro antiproliferative effects in feline primary (CAT-MT and FCMp) and metastatic (FMCm) cell lines, as well as a conserved cell death mechanism, by apoptosis, comparing to the human breast cancer cell line, SKBR-3. Furthermore, promising synergistic antiproliferative effects were obtained by combining both mAbs (trastuzumab plus pertuzumab), and mAbs with the TKi, lapatinib, already referred to as a valuable therapeutic protocol for humans. Additionally, mutations found in the clinical samples were never reported as inducing resistance to therapy, in breast cancer patients. Concluding, the use of anti-HER2 mAbs and combined protocols are proposed as a new targeted therapy for cats with different mammary carcinoma subtypes, suggesting that mAbs-resistant FMCs are rare. Still, more studies are needed to produce felinized anti-HER2 monoclonal antibodies, preventing cat immune reactions. Moreover, the similarities between the FMC and human breast cancer were demonstrated, reinforcing the utility of the cat as a breast cancer model.


Ovarian cancer is the most deadly gynecologic malignancy worldwide. Although the primary response to chemotherapy is high, the majority of patients will develop resistance against applied treatment. In this study, we focused on resistance to cisplatin, a first-line drug used for the treatment of ovarian cancer. The mechanism of the resistance development process is widely described, but there is a lack of information about the involvement of members of small heat shock proteins (HSPs) and their transport via exosomes. In this study, we used two cell lines: A2780 and SKOV3, and their cisplatin-resistance variants: A2780 CDDP and SKOV3 CDDP. We have shown that the expression of three small HSPs  in cisplatin-resistant cell lines differs from their sensitive counterparts. Further, we isolated exosomes and determined the small HSPs in their cargo. In A2780 WT we observed a low amount of HSPB5 and HSPB6. We did not observe the expression of small HSPs in the SKOV3 cell line in both sensitive and resistant variants. Our data suggest the involvement of small HSPs in drug resistance of ovarian cancer and their presence is not related to exosomal transport. Analysis of the biological consequences of the imbalance of small HSPs expression in cisplatin resistance needs further investigation.  Ovarian cancer (OvCa) is still one of the most challenging cancers in terms of improvement of existing therapies with the highest mortality rate among gynecologic malignancies. A few causes are responsible for the occurrence of those circumstances. Firstly, lack of effective screening based on biomarkers or on ultrasound examination and asymptomatic early stages of the disease, lead to its manifestation at advanced stages with a significantly reduced 5-year survival rate  are now intensively studied, due to their huge potential as a specific biomarker of several tumors and response to applied treatment  . Recental., 2012; Lancastal., 2012). Moreoval., 2012; Gonda eal., 2012).In terms of chemoresistance, the involvement of small HSPs and their secretion through the exosomal pathway in ovarian cancer is not a well-known phenomenon. They are identified as pro-cancer drivers and can be involved in the activation of anti-apoptotic response, cancer cell proliferation, and metastasis development  in chemoresistance in ovarian cancer, we aimed to identify and compare the expression of Hsp20 (HSPB6), Hsp22 (HSPB8), and alpha-B Crystallin (HSPB5) in two cell lines: A2780 and SKOV3, both in two variants: wild type and cisplatin-resistant. We also aimed to analyze and point out the differences between the Hsp expression profile between exosomes derived from wild-type, and cisplatin-resistant cell lines.Two ovarian cancer cell lines were used in this study: A2780 and SKOV3 . From each of these cell lines were generated cisplatin-resistant cell variants named as follows: A2780 CDDP and SKOV3 CDDP. The cell culture conditions and derivation of cisplatin-resistant cell lines were conducted and confirmed as previously described .The exosomes were isolated using serial differential centrifugations. Briefly, cells were washed twice with Dulbecco's phosphate-buffered saline  and a serum-free medium was added. After 48 hours the conditioned medium was collected and centrifuged for 10 minutes at 800 x g and then for 20 minutes at 3000 x g at 4 °C to eliminate larger vesicles and cellular debris. The supernatant was collected and stored at -80 °C until further use. After thawing, approximately 500 ml conditioned medium was filtered through a 0.2 μm membrane and concentrated using 100 kDa cut-off centrifugal filters . The concentrated conditioned medium was diluted 1:1 with DPBS and centrifuged at 120,000 x g for 90 minutes at 4 °C . The obtained exosomal pellet was resuspended in DPBS or RIPA buffer according to further analysis. The concentration of exosomes was determined using Pierce™ BCA Protein Assay Kit  according to the manufacturer's instructions.To confirm the presence of exosomal acetylcholinesterase activity in the isolated exosomes sample, 5 μg of exosomal protein was diluted in 50 μl of DPBS and mixed with 50 μl of freshly prepared reaction mixture consisted form 0.2 mM 5,5'-dithiobis(2-nitrobenzoic acid) and 2.5 mM acetylthiocholine  and transferred to 96-well plate. After 30 minutes of incubation at 37 °C, the absorbance was measured at 405 nm using a plate reader . As a control, DPBS mixed with reaction mixture was used.6 in TRI reagent . Next, 1 µg of total RNA was reverse transcribed using the iScript kit  according to the manufacturer's protocol. The cDNA was amplified in a total volume of 20 µl and diluted 20 times. Further, the analysis of expression of genes - HSPB5 , HSPB6 , HSPB8  - was analyzed using RT-qPCR. As a reference gene for determination relative expression Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene was used . The PCR reaction was carried out in CFX96 Touch Real-Time Detection System  in total 10 µl volume, which consisted of 2.5 µl cDNA, FastStart Essential DNA Probes Mix, and specific probes . RNA was isolated using Direct-zol RNA MiniPrep  followed by suspension of 1x10Total proteins were extracted from cell lines and exosomes using RIPA lysis buffer mixed with proteases inhibitor cocktail . Protein concentrations were determined using the Pierce™ BCA Protein Assay Kit kit . The procedure was performed like previously described . BrieflThe statistical analyses of gene expression and Western blot analysis (Welch's t-test) were performed using the GraphPad Prism 6 package . The data were deemed significant at p < 0.05.HSPB5 in CDDP resistant variants compared to WT  in OvCa during the development of chemoresistance, we used OvCa cell lines: A2780 and SKOV3, which represents distinct types of ovarian cancer cell lines. A2780 cell line represents the endometroid subtype with non-applied treatment. On the other hand, the SKOV3 cell line is derived from cells of a patient diagnosed with high-grade serous ovarian cancer with intrinsic resistance mechanisms to treatment. For over six months, we generated the cisplatin-resistant variants cell lines (named as follows: A2780 CDDP and SKOV3 CDDP), which were already described and characterized in a previous study . BrieflFigure 1a. In the Figure 1a. In the Figure 1a. The anaFigure 1a. The HSPFigure 1b. The anaFigure 1b. On the Figure 1b. Further, to validate observed transcriptomic changes of the expression of small HSPs between sensitive and resistant to cisplatin OvCa cell lines we performed a Western blot analysis . To confirm their presence, the activity of acetylcholinesterase (AChE) was assessed Figure 2a. The incOvCa patients despite relatively good responses to first-line treatment have a high risk of relapse and development of chemoresistant phenotype. This led to tumor recurrence in about 75 % of patients . The mein vitro model of two OvCa cell lines: A2780 and SKOV3 (sensitive and resistant to cisplatin), representing endometrioid and serous OvCa, respectively. To exclude the genetic drift during the prolonged culture to obtain stable resistance to cisplatin phenotype of OvCa cell lines, we used as control parallel cultured cells not exposed to CDDP . In theThe group of small HSPs is widely studied regarding their function in cellular processes particularly in the context of human diseases and cancer . In tervia transforming growth factor TGF-α in SKOV3.ip1 cells . In theal., 2015). Elevatal., 2015; Piccoleal., 2015). Recental., 2015). These al., 2015) it was al., 2015; Oesterral., 2015). No othvia exosomal cargo in pathophysiological events such as ischemic stroke or myocardial infarction has huge implications on their recovery . Howeveal., 2021; Yu et aal., 2021). Howeveal., 2021) revealeal., 2021). Moreoval., 2021). Our pral., 2021). One ofin vivo evaluation to receive a holistic overview of the molecular mechanisms of sHSP-dependent chemoresistance in ovarian cancer. This data discrepancy and the differences, which we obtained in our study, can only confirm how complicated and heterogenous the pathology of OvCa is. Our preliminary data and recently shown data provides insight into the possible interference of small HSPs in chemoresistance, suggesting that they may serve as a potential target to eliminate OvCa resistance platinum-based chemotherapy. We are aware that additional experiments regarding the administration of synthetic small HSPs or their elimination through siRNA or miRNA should be conducted in future studies focusing on global data analysis and This research benefited from a Greater Poland Cancer Center internal grant no.: 17/2017(160).The authors declare no conflict of interest.


S)-1-methylspermidine. It was reported previously ; 1072–81, 2013) that DFMO treatment leads to depletion of cellular thymidine pools, and that exogenous thymidine supplementation partially prevents DFMO-induced cytostasis without affecting intracellular polyamine pools in HT-29, SW480, and LoVo colorectal cancer cells. Here we show that thymidine did not prevent DFMO-induced cytostasis in DU145, LNCaP, MCF7, CaCo2, BT4C, SV40MES13, HepG2, HEK293, NIH3T3, ARPE19 or HT-29 cell lines, whereas administration of functionally active mimetic of spermidine, (S)-1-methylspermidine, did. Thus, the effect of thymidine seems to be specific only for certain cell lines. We conclude that decreased polyamine levels and possibly also distorted pools of folate-dependent metabolites mediate the anti-proliferative actions of DFMO. However, polyamines are necessary and sufficient to overcome DFMO-induced cytostasis, while thymidine is generally not.Polyamine spermidine is essential for the proliferation of eukaryotic cells. Administration of polyamine biosynthesis inhibitor α-difluoromethylornithine (DFMO) induces cytostasis that occurs in two phases; the early phase which can be reversed by spermidine, spermine, and some of their analogs, and the late phase which is characterized by practically complete depletion of cellular spermidine pool. The growth of cells at the late phase can be reversed by spermidine and by very few of its analogs, including ( Hypusine is a unique amino acid, formed to the ε-amino group of Lys-50 of eukaryotic initiation factor 5A (eIF5A) protein as a result of posttranslational modification . Spd. Spd®, Sfication . AlthougDFMO is in clinical use against stage 2 African sleeping sickness (trypanosomiasis) and facial hirsutism, and it has been extensively tested as an anticancer agent ,5. MoreoPreviously it was shown that in addition to depleting polyamine pools, DFMO treatment led to a reduction of cellular nucleoside and nucleotide pools, especially that of thymidine-5′-monophosphate (dTMP) . In addiIn the present study, we investigated whether such growth-supporting effect of thymidine is a general feature of other cell lines, or whether it is restricted to some specific cell lines, such as colon carcinoma cell lines. We found no evidence of the ability of thymidine to rescue DFMO-induced growth inhibition either in CaCo2 or HT-29 colon carcinoma cells or in any other of the eight tested cell lines.S)-1-MeSpd was synthesized as described previously [L-methionine were purchased from Sigma-Aldrich . SAM was purchased from Shanghai Kinpharm Engineering . The cell lines DU145, LNCaP, MCF7, BT4C, CaCo2, SV40MES13, HEK293, and HepG2 were obtained from American Type Culture Collection . ARPE-19 cell line was a kind gift from Prof. A. Kauppinen . HT-29 cell line was kindly provided by the Institute of Public Health and Clinical Nutrition . High-glucose DMEM and L-glutamine were from Sigma-Aldrich, heat-inactivated fetal bovine serum from Gibco , and gentamycin from Lonza .-1-MeSpd in the absence or presence of 5 mM DFMO. For testing of the natural polyamines, 10 μM Put, Spd, or Spm was used with 1 mM aminoguanidine . Cell proliferation was analyzed using resazurin staining [The cells were cultured in high-glucose Dulbecco’s Modified Eagle’s Medium supplemented with 10% heat-inactivated fetal bovine serum, 2 mM staining . The corstaining . The expThe cells grown on 6-well plates were detached using a solution containing 0.25 % trypsin and 1 mM EDTA in PBS. The washed cell pellet was mixed with sulfosalicylic acid (5% (w/v) final concentration) containing 10 μM diaminoheptane as internal standard and analyzed with HPLC , equipped with Pickering PCX 5200 post-column derivatization unit  as described earlier . The resOne-way ANOVA with Dunnett’s or Tuckey’s post hoc test was used for statistical analysis with the aid of software package GraphPad Prism v. 5.03.S)-1-MeSpd. , or in various other cell lines. The selected cell lines were human hormone-insensitive prostate cancer cells (DU145) and human hormone-sensitive prostate cancer cells (LNCaP), human breast carcinoma cells (MCF7), immortalized mouse kidney mesangial cells (SV40MES13), human retinal pigment epithelial cells (ARPE-19), rat glioma cells (BT4C), human embryonic kidney cells (HEK293), human hepatoma cells (HepG2), and mouse embryonic fibroblasts (NIH3T3). As indicated in 1-MeSpd. . We chos1-MeSpd. . ChanginIn contrast to earlier publication , we coulL-methionine  did not affect cell growth (Treatment with l growth .L-methionine treatments (S)-1-MeSpd treatment further decreased intracellular Spd and Spm levels, but the total polyamine level (Spd+Spm+MeSpd+MeSpm) was restored to control levels due to the high accumulation of the analog and its intracellularly converted metabolite, (S)-1-MeSpm. Interestingly, treatment with the combination of DFMO+SAM increased the amount of Spd and the total polyamine pool and decreased the amount of Spm  levels, which were not affected by thymidine or by eatments . (S)-1-Mt of Spm . By contt of Spm .L-methionine decarboxylase (AdoMetDC) yields decarboxylated SAM (deSAM), which is used as aminopropyl donor in the synthesis of Spd and Spm -1-MeSpd can reverse the “late phase” of cytostasis which is caused by depletion of hypusinated eIF5A. In DU145 cell culture supplemented with 5 mM DFMO, the “late phase” is achieved typically within a week [Our and others’ earlier work shows that DFMO-induced “early phase” of cytostasis can be reversed by the natural polyamines and many of their analogs, even those which are not hypusine precursors ,15. Howen a week ,15,16, wn a week . In thein a week  used DFMHere we were unable to obtain any reversal of DFMO-induced early cytostasis by using thymidine supplementation in any of the tested cell lines, even with shorter incubation time . Yet, the classical add-back experiments with natural polyamines and methylated Spd analog restored proliferation. These results indicate that reversal of DFMO-induced cytostasis by thymidine seems to be restricted only to some special cell types, such as SV480 and LoVo. It seems that it is not even specific for colon carcinoma cells in general, since we observed no growth-supporting effect of thymidine in CaCo2 colon carcinoma cells. Thymidine was even cytotoxic to CaCo2 cells, as indicated by increased LDH release . Here weSurprisingly, SAM supplementation could partially reverse DFMO-induced cytostasis in some cell lines, even though SAM alone was cytostatic. Examination of the intracellular polyamine pools revealed that the SAM+DFMO combination resulted in increased Spd and total polyamine pools and decreased Spm pool as compared to DFMO-treated cells. Therefore, increased total polyamine level could be responsible for the partial reversal of DFMO-induced cytostasis by SAM observed in some cell lines. In addition, some effects of SAM could be mediated by its degradation product 5′-deoxy-5′-methylthioadenosine (MTA). SAM is unstable in solution and is converted to MTA spontaneously, and intracellularly through the polyamine pathway . MTA hasA sufficient pool of dTMP is essential for DNA synthesis, and thus, for cellular proliferation. While putrescine supplementation restores both polyamine and thymidine pools of DFMO-treated cells, thymidine supplementation restores only the dTMP pool . Based o


The cardiovascular disease of atherosclerosis is characterised by aged vascular smooth muscle cells and compromised cell survival. Analysis of human and murine plaques highlights markers of DNA damage such as p53, Ataxia telangiectasia mutated (ATM), and defects in mitochondrial oxidative metabolism as significant observations. The antiageing protein Klotho could prolong VSMC survival in the atherosclerotic plaque and delay the consequences of plaque rupture by improving VSMC phenotype to delay heart attacks and stroke. Comparing wild-type VSMCs from an ApoE model of atherosclerosis with a flox’d Pink1 knockout of inducible mitochondrial dysfunction we show WT Pink1 is essential for normal cell viability, while Klotho mediates energetic switching which may preserve cell survival. Methods: Wild-type ApoE VSMCs were screened to identify potential drug candidates that could improve longevity without inducing cytotoxicity. The central regulator of cell metabolism AMP Kinase was used as a readout of energy homeostasis. Functional energetic switching between oxidative and glycolytic metabolism was assessed using XF24 technology. Live cell imaging was then used as a functional readout for the WT drug response, compared with Pink1 (phosphatase-and-tensin-homolog (PTEN)-induced kinase-1) knockout cells. Results: Candidate drugs were assessed to induce pACC, pAMPK, and pLKB1 before selecting Klotho for its improved ability to perform energetic switching. Klotho mediated an inverse dose-dependent effect and was able to switch between oxidative and glycolytic metabolism. Klotho mediated improved glycolytic energetics in wild-type cells which were not present in Pink1 knockout cells that model mitochondrial dysfunction. Klotho improved WT cell survival and migration, increasing proliferation and decreasing necrosis independent of effects on apoptosis. Conclusions: Klotho plays an important role in VSMC energetics which requires Pink1 to mediate energetic switching between oxidative and glycolytic metabolism. Klotho improved VSMC phenotype and, if targeted to the plaque early in the disease, could be a useful strategy to delay the effects of plaque ageing and improve VSMC survival. Cardiovascular diseases such as atherosclerosis exhibit profound ageing of the vessel wall that is linked to excess DNA damage and mitochondrial dysfunction . Indeed,Aged VSMC mitochondria appear to lose their ability to generate energy-carrying adenosine triphosphate (ATP). Intervening in oxidative metabolism required for normal cellular homeostasis is an attractive candidate to improve plaque cellular energetics. Indeed, our previous work has shown that diseased VSMCs retain their ability to energetically switch between oxidative and glycolytic metabolism. However, to date, there has been no effort to identify potential drug candidates that may mediate this switch. If a regulatable switch to aerobic glycolysis could be achieved pharmacologically, then improving VSMC survival could be a realistic goal.Klotho is a powerful antiageing hormone whose abundance naturally decreases with normal human ageing . Early tKlotho exists in three isoforms of α, β, and γ which may be membrane bound, intracellular, and secreted. Klotho is found as both a receptor and cleavable ligand  and has It also acts as glucuronidase and protects against endothelial dysfunction by regulating nitric oxide by impacting signalling pathways including the p53/p21 axis, cAMP, protein kinase C, and the Wnt signalling pathways , all impBuilding upon our previous work to characterise the role of Pink1-KO in atherosclerotic lesions, this study uses the generation of a vascular specific and inducible Pink1-KO cell line as a novel model of mitochondrial dysfunction . Here, wWe identified 10 compounds from the literature that have been shown to play important roles in cell survival, either through activation of the energy sensor AMPK or by evidence of inducing energetic switching between oxidative and glycolytic metabolism. To assess their suitability, a dose–response and cytotoxicity assay was performed A–K with Metformin (0.1–1 mM), A769662 (5–100 µM), and resveratrol 10 µM were taken forward as potential candidate drugs for an AMPK activation assay using Western blot. A–E Supp. We usedXF24 quantifies oxidative phosphorylation by measuring oxygen consumption rate (OCR) as a direct measure of complex 4 activity as the terminal electron acceptor of the mitochondrial respiratory chain, while simultaneously measuring extracellular acidification rate (ECAR) as cytoplasmic milli-pH as in index of glycolysis through glucose to lactate conversion. Drug doses for energetic assays were all 1 µM final concentration for mitochondrial inhibitors oligomycin, FCCP and rotenone, and myxothiazol, and 2-deoxy-glucose (25 mM) to competitively inhibit glucose (2.5 mM), as previously published . In vivoTo validate if these changes in metabolism lead to a functional change in cell behaviour, we performed live-cell imaging . First, we compared the control wild-type VSMCs’ response in the presence and absence of Klotho, through which we observed improved cell survival C. We theWe then compared rates of survival between wild-type and Pink1 knockout cells. We found Pink1-KO were significantly more difficult to grow in culture, with less than half the plating efficiency of WT cells A. NevertImproving vascular smooth muscle cell longevity is an important though challenging goal of delaying features of vulnerable or rupture-prone atherosclerosis. Indeed, VSMCs provide biochemical and mechanical strength to the plaque through the production of collagen and extracellular matrix, which are features lost as the plaque ages.Previously, we identified loss of mitochondrial homeostasis as key to this phenotype in human atherosclerosis . More reDespite AMPK being the proposed target for the action of these drugs, we found a wide variety of responses and therapeutic doses, ranging from mM doses for metformin to sub nM concentrations for Klotho. This broad range of responses makes interpretation and comparison particularly difficult. Therefore, we chose a more direct measure of energetic performance by measuring oxidative and glycolytic capacity. The XF24 method allows simultaneous readings of both oxygen consumption as an index of mitochondrial energetic performance in real time, as well as changes in milli-pH, to reflect rates of glycolysis. Across a range of nM doses, we validated Klotho as the most impressive candidate that suppressed VSMC oxidative metabolism and induced a reciprocal effect in enhancing glycolytic metabolism. Of note was the reverse dose–response, in which increasingly lower concentrations of Klotho had a more significant impact on oxygen consumption. This may reflect the lower and near physiological dosing of klotho.To identify if these changes in metabolism influenced cell phenotype, we first compared primary Pink1 wild-type VSMC to Klotho. We observed no significant difference in cell survival between wild-type cells in the presence or absence of Klotho, confirming that Klotho does not directly influence plating efficiency or total cell survival over 24 h in culture. However, wild-type cells incubated with Klotho (0.01 ng/mL) increased their rate of proliferation which was significant at 12 h. The noticeable trend of increased apoptosis and necrosis over 18–24 h could then be advantageous if it reflected Klotho’s restoration of clearance of defective cell types found in the plaque.p = 0.05) and necrosis (p = 0.001). These data suggest that VSMCs are heavily reliant on mitochondrial metabolism for survival, and any switch to a more glycolytic phenotype is unable to easily rescue this type of Pink1-KO induced dysfunction. However, perhaps in the absence of Pink1 which flags mitochondria for degradation, Klotho may compensate, promoting cell clearance through apoptosis or necrosis. We and others have shown that mitochondrial respiratory proteins activity is significantly affected in the most vulnerable parts of the plaque [Next, we compared Pink wildtype and Pink1-KO cell lines, both derived from ApoE knockout mice. Pink1 kinase KO was chosen as a comparator, as it has previously been shown to be unable to maintain mitochondrial homeostasis and thereby mimic some features of those found in diseased human plaque VSMCs. Using live-cell imagi-ng, we assessed survival, proliferation, apoptosis, and necrosis as an index of drug responses. Pink1-KO cells were more difficult to culture and had ~50% reduced viability from the outset. This effect appeared to be compensated by a higher rate of proliferation but also higher rates of apoptosis and necrosis. These data highlighted the severity of the defect of Pink1-KO and that failure to maintain mitochondrial homeostasis was adversely affecting the cell’s ability to be maintained in culture. Notably, we found Pink1-KO cells had an accumulation of intracellular lipid vesicles. Long-term use of hydroxytamoxifen (OHT) in vitro (the metabolised form of tamoxifen) required to induce gene knockout can promote a more lipogenic phenotype; however, our transient use in vitro for Cre activation (<24 h) is unlikely to be sufficient for this effect. It is intriguing, though highly speculative, that loss of β-oxidation and loss of lipid processing at the mitochondria may be the result of the accumulation of these lipids, but further research is required to explore these observations. Lastly, we tested if this unstable Pink1-KO phenotype could be rescued by Klotho, according to which we had shown WT VSMC can switch between oxidative and glycolytic metabolism. The addition of Klotho appeared to promote survival but block any further proliferation driving a significant increase in both apoptosis  with a serial drug dilution in DMEM. After each drug incubation period, a further 6 h incubation with 50 μL of 5 mg/mL MTT Formazan (Sigma Aldrich 57360-69-7) was added to each well, wrapped in foil, and incubated in the same way. At the end of the MTT incubation period, all media was replaced with 100 μL of 99.5% dimethyl sulphoxide (DMSO) and 50 μL of glycine buffer (0.1 M glycine and 0.1 M NaCl adjusted to 10.5 pH with 1 M NaOH). The plate’s absorbance was immediately read at 560 nm using a VictorTM X3 PerkinElmer plate reader .Drug cytotoxicity and dose–response curves were performed using a standard MTT cell viability assay . PrimaryThe XF24 Seahorse assay  was used for cell-based mitochondrial and glycolytic determination. Primary explanted murine VSMCs were seeded into a microplate with 100 μL at 15,000 cells/well. A calibration plate was prepared with 0.5 mL of XF calibrant solution. Cell plates media was exchanged with 0% serum-free Seahorse media on the day of the assay with a minimum of five technical replicates per plate were taken. All injection ports were prepared as previously described [TM, ImageJ, and Olympus CellSenS software, Southend-on-Sea, United Kingdomas previously described [Bright-field images and scratch assay were captured and scored using Olympus IX73 and BX51 microscopes using Adobe Photoshopescribed ,38.Western blots were performed as previously described ,40,41.t-tests (two-tailed distribution) for data following a Gaussian distribution for normality. Statistical significance is presented as p < 0.05 (one star), p < 0.01 (two stars) or p < 0.001 (three stars) p < 0.0001 (four stars). For multiple comparisons, a one-way analysis of variance (ANOVA) was used to determine a significant difference between two or more independent groups with Dunnett’s post hoc analysis. For two-way multiple comparisons, a two-way ANOVA was used with a Bonferroni post hoc test. All analysis was performed and plotted on GraphPad Prism (V. 9.3.1), GraphPad Software, San Diego, CA, USA.All data are presented as mean ± standard error of the mean (SEM). Significance values were calculated using Student’s paired In this study, we predicted that VSMCs would use a mix of mitochondrial and glycolytic metabolism, but as the plaque age, mitochondrial dysfunction increases. We found Klotho could have a significant role in improving VSMC phenotype, and targeting Klotho at earlier time points in disease could induce energetic switching and improve VSMC survival in the plaque. Even modest improvement in survival and proliferation would strengthen the plaque cap and could theoretically delay rupture. Using a VSMC specific Pink1-KO, we identified a highly unstable cell line in which Klotho was able to enhance cell clearance which would be predicted to be beneficial by removing defective cell types found in the plaque. Rewiring glycolysis has been attempted before, using embryonic fibroblasts and the transcription factor Lin28 in both wild-type and Pink1-KO cell lines, with limited efficacy ,43. Our


Centromere protein N (CENP-N) has been reported to be highly expressed in malignancies, but its role and mechanism in nasopharyngeal carcinoma (NPC) are unknown.Abnormal CENP-N expression from NPC microarrays of GEO database was analyzed. CENP-N expression level was confirmed in NPC tissues and cell lines. Stable CENP-N knockdown and overexpression NPC cell lines were established, and transcriptome sequencing after CENP-N knockdown was performed. In vitro and in vivo experiments were performed to test the impact of CENP-N knockdown in NPC cells. ChIP and dual luciferase reporter assays were used to verify the combination of IRF2 and CENP-N. Western blot analysis, cellular immunofluorescence, immunoprecipitation and GST pulldown assays were used to verify the combination of CENP-N and AKT.18F-FDG uptake in cancer nests and poor patient prognosis. Transcriptome sequencing after CENP-N knockdown revealed that genes with altered expression were enriched in pathways related to glucose metabolism, cell cycle regulation. CENP-N knockdown inhibited glucose metabolism, cell proliferation, cell cycling and promoted apoptosis. IRF2 is a transcription factor for CENP-N and directly promotes CENP-N expression in NPC cells. CENP-N affects the glucose metabolism, proliferation, cell cycling and apoptosis of NPC cells in vitro and in vivo through the AKT pathway. CENP-N formed a complex with AKT in NPC cells. Both an AKT inhibitor (MK-2206) and a LDHA inhibitor (GSK2837808A) blocked the effect of CENP-N overexpression on NPC cells by promoting aerobic glycolysis, proliferation, cell cycling and apoptosis resistance.CENP-N was confirmed to be aberrantly highly expressed in NPC tissues and cell lines and to be associated with high The IRF2/CENP-N/AKT axis promotes malignant biological behaviors in NPC cells by increasing aerobic glycolysis, and the IRF2/CENP-N/AKT signaling axis is expected to be a new target for NPC therapy.The online version contains supplementary material available at 10.1186/s13046-021-02191-3. Head and neck squamous cell carcinoma (HNSCC) is a general term for cancers that occur in the anatomical areas of the nasal cavity, lips, tongue, oral cavity, pharynx, and larynx and arise from the malignant transformation of mucosal epithelium . NasophaCentromere protein N (CENP-N), also called ICEN32, has an N-terminal region that recognizes CENP-A nucleosomes and a C-terminal region that binds CENP-L to form a mitophagy-associated network . The CENInterferon regulatory factor 2 (IRF2) is located on chromosome 4q35.1 and contains 12 exons. IRF2 competitively inhibits IRF1-mediated activation of the transcription of interferon α and β and other functional genes. IRF2 has been reported to inhibit the transcriptional activation of NFAT1 to regulate cell cycle progression in CD4+ T cells, influence cell differentiation and regulate apoptosis . In addiProtein kinase B (AKT) is a core member of the PI3K/AKT signaling pathway. AKT can be widely involved in the development and progression of almost all malignancies, including NPC –14. In mHere, we first identified and validated CENP-N as an oncogene associated with the promotion and progression of nasopharyngeal carcinogenesis. We sought to explore the transcription factors that influence changes in CENP-N gene expression in NPC. Our results revealed that IRF2 bound to two sites in the promoter region of CENP-N and that promoted the transcription of CENP-N. We further explored the specific molecular mechanisms by which CENP-N promotes the malignant biological behavior of NPC cells. The results indicated that the down-regulation of CENP-N significantly inhibited AKT phosphorylation at Ser473, leading to the inhibition of AKT activation. Finally, we found the IRF2/CENP-N/AKT axis promotes malignant biological behaviors in NPC cells by increasing aerobic glycolysis, the IRF2/CENP-N/AKT signaling axis is expected to be a new target for NPC therapy.2FC| ≥ 1 and adj. P < 0.05 as screening criteria. A PPI network was constructed via String database analysis and visualized using Cytoscape [Differentially expressed genes (DEGs) were analyzed in the GSE12452 and GSE53819 gene sets using |logytoscape . Surviva2 and 37 °C. NP460 cells were cultured in growth factor-containing keratinocyte serum-free medium (KSFM) [NPC cell lines  were cultured in DMEM  medium containing 1% penicillin-streptomycin and 10% FBS  at 5% COm (KSFM) .Tissue microarrays  were provided by Fanpu Biotech . NO.1501 and NO.131 were used for CENP-N analysis. NO.132 was used for IRF2 detection. Imaging data for 21 NPC patients who underwent PET/CT imaging at the PET Imaging Center of the People’s Hospital of Wuhan University were selected, and paraffin tissue sections of the corresponding patients were obtained through the Department of Pathology of the People’s Hospital of Wuhan University  according to the procedure. β-actin was used as the internal reference. The relative mRNA expression levels of target genes were calculated by the 2https://www.genenergy.cn/) for high-throughput transcriptome sequencing of multiple cell samples using the Illumina HiSeq sequencing platform. The sequence alignment results for each group of samples were further screened for differentially expressed genes (DEGs) that showed changes after treatment using DESeq2 software, with |log2FC|≧1 and P < 0.05 defined as the screening criteria for DEGs. Finally, the obtained DEGs were incorporated into the KEGG and GO database for further enrichment analysis.KEGG is a public database containing annotations for intrinsic associations of multiple gene functions . CENP-N Western blot assay was performed as previously described . The PVDMK-2206, 2-DG and GSK2837808A, used in the study, are a chemically synthesized AKT inhibitor, a glucose uptake inhibitor and a lactate dehydrogenase A (LDHA) inhibitor, respectively . The working concentration of MK-2206 was 10 μmol/L . The worCells were incubated in pyruvate-free DMEM  for 6 h prior to glucose uptake and lactate determination assays . Then, tGlucose uptake assay: The absorbance value of each sample at 505 nm was measured using an enzyme marker. Final sample cellular glucose uptake content = medium glucose content - sample glucose content.Lactate determination: The absorbance value of each sample at 530 nm was measured using an enzyme standard. Final sample lactate concentration = (sample assay OD - blank assay OD)/(standard assay OD - blank assay OD) × sample concentration .Cell Counting Kit-8  and clonogenic assays were used to evaluate cell survival and proliferation. Cells were processed and analyzed as previously described .Detailed steps are provided in a previous article , 24. TheTwenty-four 4-week-old female BALB/c nude mice  were housed in the SPF environment of the Animal Experiment Center of the People’s Hospital of Wuhan University.Detailed steps for model establishment are provided in a previous article  The tumoWhen the average maximum diameter of the tumors in a group of nude mice was ≥1.0 cm, small animal PET/CT imaging was performed . The maximum standardized uptake value (SUVmax), the mean standardized uptake value (SUVmean) and total lesion glycolysis (TLG) were analyzed. Tumor tissues for subsequent experiments were harvested from nude mice under adequate anesthesia .The experiment was carried out according to a previous description . The priThe dual luciferase reporter assay was performed as described in a previous study . The cloCells were fixed for 30 min, and an anti-CENP-N primary antibody (1:100) and anti-AKT primary antibody (1:100) were then added dropwise and incubated overnight at 4 °C. The following day, the slides were incubated with the FITC-labeled goat anti-mouse secondary antibody (1:100) for 1 h and then with the CY3-labeled goat anti-rabbit secondary antibody (1:300) for 1 h. Subsequently, nuclei were stained with DAPI for 15 min at room temperature, and the slides were then sealed with anti-fluorescence quenching solution. Staining of cell crawling was observed, and images were acquired using a confocal microscopy  to select the appropriate field of view. Then the images were analyzed with ImageJ  . An infoThe procedure for the cellular protein immunoprecipitation assay was described in a previous study . Cells wThe GST pulldown assay procedure was performed as described in a previous study . The GST−χ ± s) values. Comparisons of two independent samples were performed using a t-test; survival analysis was performed using the Kaplan-Meier method, and statistical analysis of survival data was performed using the log-rank test. GraphPad Prism 8 was used in this study to generate relevant statistical plots. For the purpose of this study, P < 0.05 was considered to indicate a statistically significant difference.All data collected in this study were analyzed using the medical statistics software SPSS 20.0, and the data are expressed as the mean ± standard deviation .Subsequently, we found that the CENP-N expression was elevated in NPC tissues compared with NPG by using immunofluorescence staining and Western blot analysis Fig. a-b. To fP < 0.05).Then, we analyzed PET/CT imaging data of 21 patients with NPC and the immunohistochemical staining results of the corresponding pathological tissue sections and found that tumor glucose metabolism was increased in the primary lesions of patients with high expression of CENP-N in NPC tissues compared with those of patients with low expression .Furthermore, we analyzed the immunohistochemical staining results of NPC tissue microarrays containing samples from 98 patients with 10-year follow-up data and found that high expression levels of CENP-N were associated with poor survival prognosis in NPC patients .Finally, the expression of CENP-N in the NPC cell lines 5-8F, SUNE-1, CNE-2Z, 6-10B and HK-1 was analyzed. The results confirmed that CENP-N was significantly high expression in several NPC cell lines .To further investigate the role of CENP-N in nasopharyngeal carcinogenesis and development, we generated CENP-N knockdown cell lines (shCENP-N1 and shCENP-N2) with two shRNA sequences in the NPC cell lines 5-8F and CNE-2Z ; both NPC cell lines exhibited reduced cell survival and proliferation after knockdown of CENP-N, and the differences were statistically significant ; both NPC cell lines showed an increase in the apoptosis rate after knockdown of CENP-N, and the difference was statistically significant ; and both NPC cell lines showed G0/G1 cell cycle arrest after knockdown of CENP-N . These results confirmed that knockdown of CENP-N significantly inhibited the malignant proliferation of NPC cells in vivo. In addition, we performed small animal PET/CT imaging on nude mice in the xenograft tumor models of both NPC cell lines . These results confirm that knockdown of CENP-N can also suppress NPC progression by altering the expression of proteins involved in glucose metabolism, cell proliferation, cell cycling and apoptosis in vivo.Finally, we performed HE staining Fig. A, immunoWe sought to explore the transcription factors that influence changes in CENP-N gene expression in NPC. Firstly, 311 transcription factors were screened by GeneCards database. And then 30 transcription factors were screened by PROMO database. Combined with venn diagram, five transcription factors  were shared as the transcription factors of CENP-N Fig. A. FinallThe dual luciferase reporter assays results showed that the relative luciferase activities in both IRF2 + pGL3-CENP-N-1 cotransfected cells and IRF2 + pGL3-CENP-N-2 cotransfected cells were significantly enhanced relative to that in IRF2 + pGL3-Basic cotransfected cells Fig. d. These In addition, it was found that CENP-N protein expression was significantly downregulated in the shIRF2 group relative to the shNC group in the NPC cell lines Fig. e. This rTo investigate whether knockdown of CENP-N in 5-8F and CNE-2Z cells can block the oncogenic effect of IRF2 on NPC cells, we established three groups of NPC cells—shNC, shIRF2 and shIRF2 + oeCENP-N—and examined the changes in glucose uptake and lactate production, cell proliferation, apoptosis and cell cycling in the three groups of cells. The results confirmed that IRF2 knockdown produce inhibitory effect on the glucose metabolism that can be reverted by introducing CENP-N in NPC cells Fig. C-D. IRF2To further investigate the molecular mechanisms involved in the regulation of malignant biological behaviors of NPC cells by CENP-N, we examined the changes in the expression and phosphorylation levels of core proteins in common signaling pathways involved in the regulation of glucose metabolism, cell proliferation, cell cycling and apoptosis. The results showed that relative to the levels in the shNC group, the levels of p-AKT S473) and AKT were decreased and those of p-JNK and p-P53 were increased in both NPC cell lines after knockdown of CENP-N, while those of p-AKT (T308) and P53 were not significantly changed. The JNK was decreased in 5-8F cells, while it was not significantly changed in CNE-2Z cells .Subsequently, we found that the AKT1, AKT2, and AKT3 mRNA levels were decreased in the shCENP-N1 and shCENP-N2 groups relative to the shNC group Fig. b. We alsThe cellular immunofluorescence staining results revealed that CENP-N colocalized with the AKT protein in 5-8F and CNE-2Z cells Fig. c. This rNext, we investigated whether the AKT inhibitor MK-2206 can block the oncogenic effect of overexpressed CENP-N on NPC cells. We also overexpressed CENP-N in 5-8F and CNE-2Z cells using a lentiviral vector Fig. A. SubseqThese results indicated that the down-regulation of CENP-N on NPC cells significantly inhibited AKT phosphorylation at Ser473, leading to the inhibition of AKT activation and downstream potential effects on AKT signaling.To understand whether CENP-N in 5-8F and CNE-2Z cells promotes cell proliferation and cell cycling and inhibits apoptosis by promoting glucose metabolism, we established three groups of NPC cells—oeControl, oeCENP-N and oeCENP-N + 2-DG —and examined these three groups of cells. Glucose uptake and lactate production, cell proliferation, apoptosis and cell cycle changes were examined. The results showed that 2-DG blocked the promotion of glucose metabolism in NPC cells induced by overexpression of CENP-N Fig. A-B. 2-DGHowever, 2-DG is a glucose uptake inhibitor. In order to investigate whether CENP-N promotes tumorigenesis mainly by promoting aerobic glucose utilization, we established three groups of NPC cells—oeControl, oeCENP-N and oeCENP-N + GSK2837808A (a specific inhibitor of LDHA)—and examined these three groups of cells. Glucose uptake and lactate production, cell proliferation, apoptosis and cell cycle changes were examined. The results showed that GSK2837808A blocked the promotion of aerobic glycolysis in NPC cells induced by overexpression of CENP-N . b Venn diagram showing the intersection of up- and downregulated genes in the GSE12452 and GSE53819 microarray datasets. c PPI networks associated with the differentially expressed genes. d Kaplan-Meier survival curves for patients stratified by the expression levels of differentially expressed genes . e Disease-free survival curves for HNSCC patients stratified by the expression levels of core genes (http://gepia.cancer-pku.cn/). f Histogram showing CENP-N expression in normal tissues (https://www.ncbi.nlm.nih.gov/gene/). g Linear plot of CENP-N expression in a pancancer dataset (http://gepia.cancer-pku.cn/).Additional file 2: Supplementary Figure 2. In vivo experimental immunohistochemical analysis. a HE staining of all four groups of tumors . b Immunohistochemical analysis of in vivo glucose metabolism-, cell proliferation-, cell cycle-, and apoptosis-related protein expression in tumors . c Statistical analysis of the immunohistochemical results for in vivo glucose metabolism-, cell proliferation-, cell cycle-, and apoptosis-related protein expression in tumors . The data are expressed as the mean ± SD values. * p < 0.05, ** p < 0.01, ***p < 0.001.Additional file 3: Supplementary Figure 3. Overexpression of CENP-N blocks the effect of IRF2 downregulation on malignant biological behaviors of NPC cells. a Combined with venn diagram, five transcription factors  were shared as the transcription factors of CENP-N. b Expression of IRF2 in NPC and NP460 cell lines. c Changes in relative cellular glucose uptake detected after downregulation of IRF2 and overexpression of CENP-N in two NPC cell lines. d Changes in cellular lactate production detected after downregulation of IRF2 and overexpression of CENP-N in two NPC cell lines. e Changes in cell viability detected by a CCK-8 assay after downregulation of IRF2 and overexpression of CENP-N in two NPC cell lines. f Changes in cell proliferation detected by a colony formation assay after downregulation of IRF2 and overexpression of CENP-N in two NPC cell lines. g Changes in apoptosis detected after downregulation of IRF2 and overexpression of CENP-N in two NPC cell lines. h Changes in the cell cycle distribution after downregulation of IRF2 and overexpression of CENP-N in 5-8F and CNE-2Z cell lines. The data are expressed as the mean ± SD values. * p < 0.05, ** p < 0.01, ***p < 0.001.Additional file 4: Supplementary Figure 4. Changes in AKT S473 phosphorylation in nude mouse tumor tissues. a p-AKT (S473) immunofluorescence staining in tumor tissue in vivo . b p-AKT (S473) immunohistochemical staining in tumor tissue in vivo . c p-AKT (S473) protein expression in tumor tissue in vivo was detected using WB.Additional file 5: Supplementary Figure 5. AKT inhibitors block the effect of CENP-N overexpression on malignant biological behaviors of NPC cells. a Changes in the relative cellular glucose uptake after overexpression of CENP-N or treatment with an AKT inhibitor in two NPC cell lines. b Changes in cellular lactate production after overexpression of CENP-N or treatment with an AKT inhibitor in two NPC cell lines. c Changes in the viability of cells as detected by a CCK-8 assay after overexpression of CENP-N or treatment with an AKT inhibitor in two NPC cell lines. d Changes in the proliferation capacity of cells as detected by a colony formation assay after overexpression of CENP-N or treatment with an AKT inhibitor in two NPC cell lines. e Changes in the percentage of apoptotic cells detected after overexpression of CENP-N or treatment with an AKT inhibitor in two NPC cell lines. f Changes in the cell cycle distribution after overexpression of CENP-N or treatment with an AKT inhibitor in 5-8F and CNE-2Z cell lines. The data are expressed as the mean ± SD values. * p < 0.05, ** p < 0.01, ***p < 0.001.Additional file 6: Supplementary Figure 6. CENP-N promotes cell proliferation, cell cycling and apoptosis by increasing glucose metabolism in NPC cells. a Changes in relative glucose uptake in two NPC cell lines were detected after CENP-N overexpression and 2-DG treatment. b Changes in lactic acid production in two NPC cell lines were detected after CENP-N overexpression and 2-DG treatment. c A CCK-8 assay was used to detect changes in cell viability after CENP-N overexpression and 2-DG treatment in two NPC cell lines. d Clone formation assay was used to detect the changes in cell proliferation after CENP-N overexpression and 2-DG treatment in two NPC cell lines. e The percentage of apoptotic cells was determined in two NPC cell lines after CENP-N overexpression and 2-DG treatment. f Changes in the cell cycle distribution in two NPC cell lines after CENP-N overexpression and 2-DG treatment. The data are shown as the mean ± SD values. * p < 0.05, ** p < 0.01, ***p < 0.001.Additional file 7: Supplementary Table 1. The website addresses of datasets. Supplementary Table 2. The general characteristics of NPC patients with PET/CT imaging data. Supplementary Table 3. The references for all antibodies used. Supplementary Table 4. List of primers used for qRT-PCR analysis. Supplementary Table 5. List of primers used for ChIP analysis. Supplementary Table 6. Tissue microarray analysis of CENP-N expression.


MDM2 and CDK4. Although systemic chemotherapy is recommended for unresectable or metastatic cases, DDLPS is insensitive to conventional chemotherapy, leading to an unfavorable prognosis. Therefore, novel treatment methods are urgently required. Patient-derived cell lines are essential in preclinical studies. Recently, large-scale screening studies using a number of cell lines have been actively conducted for the development of new therapeutic drugs. However, the DDLPS cell line cannot be obtained from public cell banks owing to its rarity, hindering screening studies. As such, novel DDLPS cell lines need to be established. Accordingly, this study aimed to establish a novel DDLPS cell line from surgical specimens. The cell line was named NCC-DDLPS4-C1. NCC-DDLPS4-C1 cells retained copy number alterations corresponding to the original tumors. Further, the cells demonstrated constant growth, spheroid formation, and equivalent invasiveness to MG63 osteosarcoma cells. We also conducted drug screening and integrated the results with those of the previously reported DDLPS cell lines. Consequently, we identified the histone deacetylase inhibitor romidepsin as a novel candidate drug. In conclusion, the NCC-DDLPS4-C1 cell line is a useful tool for the basic study of DDLPS.Dedifferentiated liposarcoma (DDLPS) is a highly malignant sarcoma characterized by the co-amplification of  MDM2 and CDK4 at 12q13-15 and by ALT/WDLPS [HMGA2, TSPAN31, and CPM, have also been identified in DDLPS [Dedifferentiated liposarcoma (DDLPS) is a typical non-lipogenic sarcoma that develops from an atypical lipomatous tumor/well-differentiated liposarcoma (ALT/WDLPS) . DDLPS uLT/WDLPS . In addiin DDLPS ,7. Copy in DDLPS ,9,10,11.Although complete resection is the primary modality, it is often difficult to achieve in cases involving the retroperitoneum because of the adjacent blood vessels or organs, leading to poorer prognosis than in cases arising in the extremities ,12. MoreIn vitro studies using patient-derived cell lines are essential in preclinical studies. Short-term cultured cell lines preserve the genomic features of the original tumor ,22,23. RThus, this study aimed to establish a novel DDLPS cell line from surgical specimens. In addition to characterization of the cell line, we attempted to identify novel therapeutic candidates by integrating the results of drug screening for NCC-DDLPS1-C1 , NCC-DDLMDM2 amplification using fluorescence in situ hybridization (FISH)  with the chief complaint of a rapidly growing mass in the left abdomen and radiating pain in the left lateral thigh. Computed tomography (CT) and needle biopsy suggested malignancy, and thus, wide resection was performed. The results indicated dedifferentiated liposarcoma. At 2 years and 2 months postoperative, CT detected local recurrence in the left retroperitoneum a. Given n (FISH) d. A yearA part of the resected tumor at re-operation was used to establish the cell line described in this study. The cell line was named NCC-DDLPS4-C1.Pathological examination was performed on 4 μm-thick sections from a paraffin-embedded tumor specimen. The deparaffinized sections were stained with hematoxylin and eosin (H&E) and assessed by an expert pathologist.MDM2 amplification was detected using the ZytoLight SPEC MDM2/CEN 12 Dual Color Probe . The hybridized images were obtained using the Metafer Slide Scanning Platform .FISH analysis was also performed on 4 µm-thick sections of the tumor specimen. 2. The cells that developed from the tumor specimens were maintained for more than 6 months under tissue culture conditions and were passaged more than 25 times.The surgically resected tumor was first stored in CELLBANKER  at −80 °C. The tumor was stored for 1 year and 11 months before culturing. At the time of primary culture, the cryopreserved tumor specimen was thawed and divided into small pieces with sterile scissors. The tumor specimens were cultured in DMEM/F12  supplemented with 5% heat-inactivated fetal bovine serum (Gibco), 100 μg/mL penicillin and 100 μg/mL streptomycin , 0.4 µg/mL hydrocortisone , 5 ng/mL EGF (Sigma-Aldrich), 10 ng/mL bFGF (Sigma-Aldrich), 5 µg/mL insulin (Sigma-Aldrich), and 10 µM Y-27632  at 37 °C in a humidified atmosphere with 5% COShort tandem repeats (STRs) of the established cell line were examined at 10 loci using the GenePrint 10 system . Briefly, genomic DNA was extracted from the established cell line and the corresponding tumor tissue using AllPrep DNA/RNA Mini kits . STRs were amplified using the obtained DNA (10 ng). PCR products were analyzed using a 3500xL Genetic Analyzer . The obtained data were analyzed using the GeneMapper software (Applied Biosystems). Finally, the STR profiles were examined using a cell line database, Cellosaurus , to checMycoplasma contamination was examined using the e-Myco Mycoplasma PCR Detection Kit . Extracted cellular DNA (50 ng) was used for PCR. The DNA sequence unique to mycoplasma was amplified. The amplified DNA was electrophoresed, and the gel was stained with Midori Green Advance stain . Finally, the images were obtained using the Amersham Imager 600 system .http://www.R-project.org, accessed on 26 September 2021) and DNAcopy package version 1.64.0 . Chromosome regions with copy numbers >3 or <1 were regarded as amplifications and losses, respectively. Genes that exhibited CNAs were annotated using the biomaRt package version 2.46.0 (Bioconductor) and the GRCh 37 assembly in Ensembl .Single nucleotide polymorphism (SNP) array analysis was performed using the Infinium OmniExpressExome-8 v1.4 BeadChip . Briefly, the extracted genomic DNA of the established cell line and the corresponding tumor tissue were amplified and hybridized onto array slides in an iScan system (Illumina). Log R ratios were calculated using the Genome Studio 2011.1 (Illumina) and analyzed using the R version 4.0.3  as a positive control and normal human foreskin fibroblast (HFF) cell line  as a negative control according to a previously reported procedure . First, 4) were seeded in 24-well culture plates on day 0. The number of cells was assessed based on the absorbance at 450 nm after treatment with the Cell Counting Kit-8. Absorbance was measured every other day until day 4. The doubling time was calculated based on the growth curve obtained.Cell proliferation assays were performed using the Cell Counting Kit-8 . The cells (1 × 105) were seeded in a plate and incubated for 3 days. Spherical colonies were embedded in paraffin and fixed with iPGell . Then, paraffin sections were prepared for H&E staining.Spheroid formation was assessed using 96-well round-bottom ultra-low attachment microplates . Cells (1 × 104) suspended in serum-free DMEM/F12 medium were seeded in the upper chamber coated with BD Matrigel matrix . The culture medium, which was used to maintain the cells, was added to the lower chamber. Then, the electronic sensors detected the cells that migrated from the upper chamber to the lower chamber. Measurements were taken every 15 min for 72 h. In this experiment, MG63 osteosarcoma cells  were used as controls [Invasion assays were performed using a real-time cell analyzer . Briefly, the cells (4 × 10controls .6) were suspended in a 100 μL 1:1 mixture of BD Matrigel matrix and D-PBS (-)  and injected subcutaneously into four locations of female BALB/c nude mice . Tumor size was measured weekly, and the volume was estimated according to the following formula: volume = (length × width2)/2. After 2 months, the tumors were surgically excised and prepared for H&E staining.Animal experiments were conducted following the guidelines of the Institute for Laboratory Animal Research, National Cancer Center Research Institute. Briefly, the cells (1 × 104) were seeded in a 384-well plate and incubated for 1 d. On the second day, 10 µM of each drug was added. On the fifth day, cell proliferation was measured based on the absorbance at 450 nm using a Cell Counting Kit-8. Cell viability was calculated by comparing with the DMSO control. The obtained data were integrated with the results of NCC-DDLPS1-C1 [https://cran.r-project.org, accessed on 26 September 2021).Drug screening tests were performed using 197 drugs, including FDA-approved anticancer agents  value was calculated for the drugs that exhibited remarkable antiproliferative effects in drug screening and standard treatment drugs. In short, the selected drugs were dispensed in the cells seeded as the preceding procedure at 10 different concentrations from 0.1–100,000 nM. Based on the cell viabilities calculated in the same way, IC50 values were determined using GraphPad Prism 9.1.1 .Subsequently, the half-maximal inhibitory concentration (ICThe NCC-DDLPS4-C1 cell line was authenticated by analyzing the STR at 10 loci . The STRMDM2 and CDK4 were co-amplified, which is characteristic of DDLPS. In addition to MDM2 and CDK4, multiple genes with CNAs were observed.SNP array analysis revealed that NCC-DDLPS4-C1 cells harbored multiple CNAs (amplification: 11p14, 12q13-15, 12q21-23, and 15q22. Losses: 3q29, 8p23, 11q23, 15q21, 18q21, and 19q13; MDM2 and CDK4 by SNP array, we confirmed the high expression of MDM2 and CDK4 by Western blotting (In addition to the amplification of blotting a. These Microscopically, NCC-DDLPS4-C1 cells comprised elongated spindle cells under culture conditions a,b. UsinAmong the four locations in which NCC-DDLPS4-C1 cells were injected in BALB/c nude mice, tumor growth was observed in one location . The tumThe antiproliferative effects of 197 drugs on NCC-DDLPS4-C1 cells were examined. The cell viability after treatment with each drug at a concentration of 10 µM is shown in 50 values of NCC-DDLPS4-C1 were determined for the drugs that were used in the screening study. The IC50 values of NCC-DDLPS4-C1, NCC-DDLPS1-C1, NCC-DDLPS2-C1, and NCC-DDLPS3-C1, which we previously reported, are listed in The ICDDLPS is a highly malignant sarcoma with an unfavorable prognosis. Although systemic chemotherapy is recommended for unresectable or metastatic cases, conventional chemotherapy has no overall survival benefit . PreclinWe established the NCC-DDLPS4-C1 cell line from the recurrent tumor of elderly patients with DDLPS. In addition to the typical elderly age of patients, another common feature of DDLPS is retroperitoneal origin. Thus, the NCC-DDLPS4-C1 cell line established from an elderly DDLPS patient was useful for understanding the typical nature of DDLPS.MDM2 and CDK4, multiple gene amplifications were observed in chromosome 12q13-15 of the NCC-DDLPS4-C1 cell line. The co-amplification of HMGA2, TSPAN31, and YEATS4 with MDM2 is frequently observed in DDLPS and is involved in tumor development [CPM knockdown resulted in inhibition of DDLPS cell proliferation, migration, and invasion, suggesting an association with malignant behavior [SLC35E3, MDM2, and CPM is also involved in the development of DDLPS [FRS2 is also frequently observed in DDLPS. FRS2 is related to the FGF receptor signaling pathway, suggesting that it may be a therapeutic target [The NCC-DDLPS4-C1 cell line harbored CNAs similar to those of the original tumor, indicating that the NCC-DDLPS4-C1 cell line retained its genomic characteristics under the culturing conditions. In addition to the co-amplification of elopment ,37. CPM behavior . Simultaof DDLPS . The ampc target ,40. Excec target , NCC-DDLc target . Loss ofc target . HoweverNCC-DDLPS4-C1 cells demonstrated typical spindle cell morphology under culture conditions and the capability of spheroid formation. The co-expression of MDM2 and CDK4 also supported that the NCC-DDLPS4-C1 cell line maintained the characteristics of DDLPS. Compared to the DDLPS cell lines we previously reported ,31,32, tDrug screening identified multiple novel therapeutic candidates. Among them, the HDAC inhibitor romidepsin showed equivalent to superior antiproliferative effects on NCC-DDLPS4-C1 cells, along with NCC-DDLPS1-C1 , NCC-DDLA strength of this study is that a novel DDLPS cell line, NCC-DDLPS4-C1, was established. Further, novel therapeutic candidates, such as romidepsin, were identified. However, this study also has some limitations. Although this study is one of the largest studies to summarize the results of drug screening for DDLPS, the number of cell lines is still lower than in other common cancers. For example, it was reported that 30 cell lines are needed in a drug screening test to detect novel therapeutic drugs at a statistical power of 70% . In addiWe successfully established a novel DDLPS cell line, NCC-DDLPS4-C1. NCC-DDLPS4-C1 cells retained the characteristics of the original tumors. We also found that the HDAC inhibitor romidepsin has a remarkable antiproliferative effect on NCC-DDLPS4-C1 cells. However, the number of DDLPS cell lines remains insufficient. More DDLPS cell lines need to be established to reveal the complexity and diversity of DDLPS. The established cell line should be broadly shared in the research community to promote the study of rare malignancies such as DDLPS.


Thus, the objective of this study is to evaluate and compare the cytotoxicity and antiproliferative effects of cis- and trans-TMS in MCF-7 and its normal counterpart MCF-10A. Both compounds were cytotoxic, genotoxic, and induced G2-M accumulation and cell death in the two cell lines. These results suggested that the genotoxicity of cis- and trans-TMS is involved in the reduction of cellular proliferation of MCF-7 and MCF-10A cells, but notably, such antiproliferative effects are more pronounced for cis- than trans-TMS.Stilbenes are a class of natural compounds with a wide variety of biological effects, such as antitumor activity. The best-known stilbene is resveratrol, whose clinical application is limited due to its low bioavailability. Methoxylated derivatives of this stilbene, including  Natural products are an important source of bioactive agents of diverse structures and biological activities. They represent the most abundant reserve of molecules for the development of drugs in clinical use . ConsideThe stilbenes are among the natural compounds with great pharmacological potential. The most studied stilbene is resveratrol, a polyphenolic compound found in a wide variety of plant species such as grapes, blueberries, among others . Studiesin vivo studies have shown that issues such as poor bioavailability due to quick metabolism can hamper the clinical use of resveratrol; this problem can be overcome using polymeric and lipid nanoparticles to improve the oral bioavailability of resveratrol (Z)-1,3-dimethoxy-5-(4-methoxystyryl)benzene (cis-TMS) and (E)-1,3-dimethoxy-5-(4-methoxystyryl)benzene (trans-TMS)  and its standard counterpart (MCF-10A).Despite these promising effects, veratrol . Some stveratrol . Therefoveratrol . Structuveratrol . Thus, tans-TMS) . Some reans-TMS) . Cis-TMS effects . As well effects . Despiten-butyllithium  and the resulting solution was stirred under inert atmosphere for 2 h. A solution of aldehyde  in THF was added dropwise, and the mixture was stirred for 12 h at room temperature. The resulting suspension was poured into water and extracted with dichloromethane. The organic phase was combined and dried over MgSO4, and concentrated under reduced pressure. The crude product was purified through automated flash purification elution with hexane/ethyl acetate.The stilbenes were synthesized as previously published . ShortlyTrans-TMS. 1H NMR: δ 3.83 ; 6.37 ; 6.65 ; 6.87-6.89 ; 6.92 ; 7.04 ; 7.45 . 13C NMR : δ 55.2 (3C), 99.7, 104.2 (2C), 114.2 (2C), 126.5, 127.8 (2C), 129, 130, 139.6, 159.4, 161.0 (2C). LC-MS m/z 270.19. (M+H)+.Cis-TMS. 1H NMR: δ 3.71 ; 3.81 ; 6.36 ; 6.47-6.50 ; 6.57 ; 6.81 ; 7.26 . 13C NMR: δ 55.2 (3C), 99.6, 106.6 (2C), 113.5 (2C), 128.7, 129.5, 130.2, 130.3 (2C), 139.5, 158.7, 160.5 (2C). LC-MS m/z 271.136. (M+H)+.2 or multiple well plates when necessary. The cells were incubated at 37 °C under an atmosphere saturated with 5% of CO2. MCF-7 and MCF-10A cells, obtained from Cell Bank of Rio de Janeiro, were cultured in complete medium containing DMEM+HAMF10 medium , supplemented with 10% of fetal bovine serum and antibiotic mixture penicillin/streptomycin (10 mL/L) plus kanamycin sulfate (10 mg/L) using culture bottles of 25 mmcis-TMS and trans-TMS were synthesized as previously reported , 0.5 µM of doxorubicin, or 0.1% of DMSO. Doxorubicin and DMSO were positive and vehicle control, respectively. The cells were incubated for an additional 24 h and, the plate was washed with phosphate buffer saline (PBS 1x). The plate was then incubated with DMEM without phenol red plus the XTT/electron solution for 4 h. Total absorbance was measured at 492 and 690 nm. The number of viable cells was directly proportional to the absorbance and the percentage was compared with the negative control.Cytotoxicity assay used the Cell Proliferation Kit (Roche Applied Science). On a 96 wells plate, 10trans-TMS  and cis-TMS  for 24 h. After this period, each well was washed with PBS 1x, completed with medium, and allowed to grow for 7-14 days, when the colonies were visible. The cells were fixed with a mixture of methanol/acetic acid/water  for 30 min and stained with Giemsa/Sörensen phosphate buffer  for 15 to 30 min. The colonies were counted using a stereomicroscope, and the cell survival fraction was calculated as a percentage compared to the negative control.The clonogenic assay was according to trans-TMS and cis-TMS for four hours. After treatments, 20 µL of cell suspension was mixed with 200 µL of low melting point agarose (0.5%). This cell suspension/agarose mixture was poured on the previously coated slides with agarose (1.5%) and covered with a coverslip. After solidification, the coverslip was removed, and the slides were placed in a cold lysis solution  for at least two hours. Then, they were incubated in alkaline buffer  at 4 °C for 20 min and submitted to electrophoresis for 30 min at 25 V 300 mA. The slides were incubated in a neutralization buffer for 15 min  and fixed with 15% CCl3COOH, 5% ZnSO4 7 H2O and 5% C3H8O3 for 10 min. All slides were stained using silver nitrate as described by The alkaline comet assay evaluated the genotoxicity. Ten thousand cells/well were seeded in 12 wells plates, and after 24 h, treated with different concentrations of 4 cells/wells were seeded in 12 wells plate, and after 24 h, the cells were treated for an additional 24 h with 2.5 µM of cis-TMS or 10 µM of trans-TMS. The cells were harvested and stained with propidium iodide to determine the different phases of the cell cycle by flow cytometry using the Flow Cytometry BD FACSCanto .For cell cycle kinetics, 5x104 cells/well were seeded in 12 wells plate. After 24 h, the cells were treated with 2.5 µM of cis-TMS or 10 µM of trans-TMS and incubated for 24 h. Then, the cells were harvested and processed according to the manufacturer’s instructions using the Dead Cells Apoptosis Kit . The apoptotic cell frequency was determined using the Guava® EasyCyte™ . By this procedure, early apoptotic cells are Annexin V-positive and PI-negative (Annexin V-FITC+/PI−), whereas late (end-stage) apoptotic cells are Annexin V/PI-double-positive (Annexin V-FITC+/PI+)  .All statistical analyses were performed using GraphPad Prism 5.0 software. Analysis of variance (ANOVA), followed by Bonferroni post-test, compared the experimental groups. Statistically significant values were those with p ≤ 0.05, and the results were reported as means and standard deviations (SD) of three independent experimental samples. Negative control (NC) and vehicle control (VC) did not exhibit statistical differences; therefore, treatments were compared to the vehicle (VC).cis- and trans-TMS concentrations. Both compounds caused a significant reduction of cell viability at all tested concentrations in both cell lines (p < 0.0001 vs. VC). Both substances showed concentration-dependent cytotoxic activity and were not selective between MCF-7 and MCF-10A cells since no statistical differences were detected between both cell lines. The calculated inhibitory concentration (IC50) was 42.2 and 59.5 µM for cis- and trans-TMS in MCF-7 and, 16.2 µM and 45.7 µM in MCF-10A for cis- and trans-TMS, respectively.cis-TMS, while for the trans-TMS, the concentrations of 2.5 µM, 5 µM, and 10 µM were selected. Treatment of the cells with cis-TMS significantly reduced the survival fraction of MCF-7 cells in concentrations higher than 2.5 µM (p < 0.001)   . In MCF-< 0.001) . Treatmell lines .cis- or trans-TMS in both cell lines  and MCF-10A (p < 0.05) (trans-TMS at 5 and 10 µM increased the DNA damage score only in MCF-10A cells (p < 0.05)  , while t < 0.05) .cis- or 10 µM of trans-TMS. The treatment with cis- or trans-TMS significantly reduced the frequency of G1 cells and notably increased the frequency of G2-M cells in both cell lines compared to the negative control (p < 0.05). No statistical differences in response to the treatments were observed comparing the MCF-7 and MCF-10A cells.cis- or trans-TMS. The frequency of apoptosis .In the present study, the antiproliferative effects of two stilbene derivatives, cis- and trans-TMS reduced the cellular viability of both cell lines, as demonstrated by the XTT assay. The calculated IC50 revealed higher cytotoxicity of cis- than trans-TMS. However, while relative IC50 values may have predictive value in comparing a series of compounds with a similar mode of action, absolute IC50 values do not predict cell survival (cis- or trans-TMS in both cell lines in the present study. Although the value of short-term cytotoxicity assays is based on tetrazolium salts to reveal the fraction of still metabolically active cells, these non-clonogenic assays may underestimate the cytotoxicity of a compound. In addition, these assays can overestimate the cytotoxicity by not consider cells with reversible damage or resistance to the cytotoxic agent tested (The treatment with survival . This ist tested . Loss oft tested . Therefot tested .cis-TMS inhibited the colony formation, while trans-TMS did not reduce the survival fraction to statistically significant levels. The reduction of the survival fraction after cis-TMS treatment could be related to its genotoxicity. In the presence of DNA damage that cannot be repaired, p53 protein may activate the DNA damage response pathway modulating the cell cycle progression or triggering apoptosis to prevent the replication of damaged chromosomes (cis- and trans-TMS increased the extension of DNA damage in both cell lines, but while the cis- was genotoxic to both cell lines, the trans-TMS increased the DNA damage score only in MCF-10A and in concentrations two or four times higher than the cis. According to our results, even low concentrations of the omosomes . Both cicis-TMS and trans-TMS displayed cytotoxic and genotoxic effects, the ability of these two substances to modulate the cell cycle kinetics and induce cell death was investigated. Both stilbenes caused an accumulation of cells in the G2 phase and increased the frequency of apoptosis in MCF-7 and MCF-10A. The accumulation of both cell lines in the G2-M phase may be related to the genotoxicity of the substances since the induction of DNA damage during the S phase can lead to the G2-M cell cycle arrest and subsequent death by apoptosis. Treatment of hepatoma-derived cell lines with 1 µM of cis-TMS during 48 h induced accumulation in the G2-M phase with a concomitant reduction in G1 and S phases (Cip/Waf1 expression. Trans-TMS at 80 µM also stalled HCT116 cancer cells in G2-M with subsequent cell death (Since both S phases . These ell death . There icis- and trans-TMS induced apoptosis in both cell lines. Apoptosis can occur in response to different situations, including after cell damage by genotoxicity caused by chemotherapeutic agents (cis- and trans-TMS are related to cell cycle arrest and apoptosis. Our results demonstrated that c agents . Our datcis- and trans-TMS are not selective between MCF-7 and MCF-10A and that the cellular response to cis-TMS occurs in concentrations four times lower than trans-TMS. Cis- and trans-TMS are geometric isomers. They are non-superimposable, non-mirror images of each other. They result from the restricted rotation of a carbon-carbon bond. The two enantiomers of a chiral drug may differ significantly in their bioavailability, rate of metabolism, metabolites, excretion, potency, selectivity for receptors, transporters, and enzymes, and toxicity, leading to the choice for a single-enantiomer drug with more straightforward and more selective pharmacologic profiles, improved therapeutic indices and simpler pharmacokinetics (When considering the overall results of the present study it is clear that kinetics . cis-TMS.In conclusion, the present results suggest that the genotoxicity of both compounds may be involved in the antiproliferative effects observed in MCF-7 and MCF-10A cell lines. However, this effect is considerably more pronounced for