EC50 value in PANC-1 cell line dropped from 4.04 ± 0.046 to 1.68 ± 0.004 µM. The ICPD47 and ICPD62 under the same conditions could act synergistically with GEM, 5-FU and DOX and is worth of further investigations, and studies of synergistic effect is a promising path for more efficient anticancer therapies.Heat shock protein 90 (Hsp90) is a widely investigated target for anticancer therapy. The experimental Hsp90 inhibitors ICPD47 and ICPD62 demonstrated anticancer activity against colorectal, osteosarcoma and cervical cancer cell lines. However, their anticancer activity has not been investigated against pancreatic cancer cell lines yet, and there are no data about synergistic activity of these compounds in combination with clinically used anticancer agents. Pancreatic cancer cell lines, MIA PaCa-2 and PANC-1 were exposed to ICPD47 and ICPD62 alone and in combinations with antimetabolites gemcitabine (GEM), 5-fluorouracil (5-FU) and topoisomerase inhibitor doxorubicin (DOX). Effects on cell viability were determined by MTT assay. The synergistic activity was evaluated using Chou-Talalay method. Also, 3D cell cultures were formed using 3D Bioprinting method and the activity of each compound and their combinations was examined by measuring the size change of spheroids. The strongest synergistic activities were determined in combinations using all ratios of ICPD47 with GEM and ICPD62 with GEM in MIA PaCa-2 cell line (combination index <0.5). The combinations of ICPD47 with 5-FU and ICPD47 with GEM in a ratio of 1:5 showed the greatest effect on tumour spheroid growth in both cell lines. The ICPD47 in combination with mild hyperthermia showed significant results, where the  In addition, the increasing resistance of pancreatic cancer to available chemotherapy is a serious problem.Pancreatic cancer is one of the most aggressive and deadliest cancer types. After diagnosis, the patients have a 5-year survival rate of only 8.5%. Such a low survival rate is mainly because the disease is diagnosed at late stages and it progresses fast to lymphatic system and local organs3. Therefore, there is a huge need for novel therapeutic agents that could act on multiple targets and enhance the success of pancreatic cancer therapy.There are seventeen registered drugs for pancreatic cancer chemotherapy, but statistics show that the current treatment is not effective. Recent studies show, that the resistance for 5-fluorouracil (5-FU) and gemcitabine (GEM) is increasing5. Since this molecule regulates multiple signalling cascades, the inhibition of Hsp90 has a suppressive effect on many client proteins and biochemical pathways5. Because of this characteristic and the fact that in cancer tissues Hsp90 is expressed in higher levels, the inhibition of Hsp90 can overcome the redundancies of signals and drug resistance in many cancers5. Recent scientific and clinical data show that Hsp90 inhibitors strongly affect cancer cell survival and using them in combinations with anticancer agents, it is possible to postpone cell resistance to chemotherapy7.Heat shock protein 90 (Hsp90) is a chaperone protein of 90 kDa. Hsp90 is required for the folding and stability of proteins, which are critical for cell growth, differentiation and survival8. To determine the combination effect, the Chou-Talalay method is often used, when the combination index (CI) is calculated. This method is the most accurate to evaluate the type of combinatorial effect of different compounds. CI values below 1 (CI < 1) describe synergistic effect, values equal to 1 (CI = 1) describe additive effect, and values over 1 (CI > 1) show antagonistic effect of the compounds8.Combinatorial chemotherapy is often used for the treatment of cancers. Synergistic effect of different drugs in combinations can increase its therapeutic effect, decrease dosage and toxicity, compared to individual drugsP values, rather than Chou-Talalay method, when CI is calculated; (b) using randomly chosen concentrations of compounds in the combination instead of using constant EC50 ratios of compounds in the combination8. Some studies have shown that the expression of Hsp90 in pancreatic cancer tissue and in tissues adjacent to cancer is higher up to five fold, compared to healthy pancreatic tissue10. With this study, we wanted to obtain new fundamental insights on the effects of Hsp90 inhibitors in combinations with already used anticancer agents  against pancreatic cancer cell lines. Therefore, for the first time, we tried to determine the combination effect of experimental resorcinol-like Hsp90 inhibitors (ICPD47 and ICPD62) and clinically used anticancer agents in two-dimensional (2D) and three-dimensional (3D) models of different pancreatic cancer cell lines. In addition, we aimed to determine whether a mild hyperthermia could increase the activity of Hsp90 inhibitors.There are many scientific manuscripts published on the combinations of Hsp90 inhibitors and anticancer agents in various cancers except for pancreatic cancer. Never the less, credible data on synergistic activity of Hsp90 inhibitors and anticancer agents are limited or falsely determined. The main mistakes in determining the combinatorial effects of the drugs are: (a) using statistical analysis and calculating p < 0.05). The ICPD62 had higher effect on cell viability than ICPD47, almost nine times in MIA PaCa-2 cell line, half maximal effective concentration (EC50) values of ICPD62 and ICPD47 after 72 h were 0.45 ± 0.06 µM and 3.91 ± 0.25 µM. And in PANC-1 cell line ICPD62 inhibited cell viability six times stronger than ICPD47, as EC50 values of ICPD62 and ICPD47 after 72 h were 0.41 ± 0.08 µM and 2.38 ± 0.25 µM .Hsp90 inhibitors and anticancer agents demonstrated different effects on cell viability Fig. . All comSimilar to DOX, both Hsp90 inhibitors showed cytotoxic activity, as they totally inhibited cell viability in higher concentrations Fig. . The GEMCI value, when fraction affected (fa) equal to 0.5, was 0.16 ± 0.05) in ratio 1:5 in MIA PaCa-2 cell line  in PANC-1 cell line  in all concentrations (fa range from 0 to 1) in MIA PaCa-2 cell line , PANC-1 cells formed relatively small and compact spheroids (401 ± 13.8 μm in diameter) Fig. . The effp < 0.05), and 5-FU reduced the size of the spheroids by 1.5 times (p < 0.05) . The least effective compounds were ICPD62 and DOX, as they reduced the size of the spheroid by 1.17 and 1.25 times respectively  Fig. . Howeverp < 0.05) and 1.35 (p < 0.05) times smaller compared to control group. However, the difference between the effects of the combination and GEM alone was not significant (p > 0.05). ICPD62, DOX and 5-FU had almost no effect on the growth of PANC-1 spheroids  and in MIA PaCa-2 - 1.2 times, from 3.71 ± 0.01 to 2.48 ± 1.65 µM (p > 0.05) , and in PANC-1 - 1.8 times from 0.39 ± 0.01 to 0.22 ± 0.02 µM, but the results were not of exceptional significance (p > 0.05)  Fig. . ICPD62 05) Fig. .Figure 8et al. study, where ICPD62 showed almost 10 times higher activity than ICPD47 in U2OS and HeLa cell lines11. Such results can be explained by the differences in compound structure12.In our study, ICPD62 showed from 6 to 9 times greater effect on cell viability than ICPD47, in PANC-1 and MIA PaCa-2 cell lines, respectively. Similar results were demonstrated in Cikotiene 14. Also it is possible, that some antimetabolites, such as cisplatin, may inhibit Hsp90 by binding to its’ C-domain, and therefore using it in combination with Hsp90 inhibitors can enhance the inhibition of Hsp9015. Kaiser et al. in their study determined that DOX in combination with resorcinol-like Hsp90 inhibitor could have synergistic effect on cell viability, at higher concentrations of compounds when higher concentrations of compounds are used. However, due to variable conditions, such as incubation plan and time with individual compounds, the effect can vary from synergism to antagonism16.The strongest synergistic activity was determined by using combinations of Hsp90 inhibitors with GEM and 5-FU. According to different studies of Fallahi-Sichani and Arlander, the Hsp90 inhibitors may slow down the cell metabolism, and therefore making them more sensitive to antimetabolitesEC50 ratio, can have different effect, even though, both inhibitors have the same mechanism of action. It is possible that such differences in action can be influenced by different structures and activities of both compounds. Lee et al. in their study determined synergistic activity of combinations of Hsp90 inhibitor NVP-AUY922 and 5-FU against gastric cancer cells17. Researchers explained that NVP-AUY922 inhibited the expression of thymidylate synthase (TS), which is the main reason of increased resistance of cancer cells to 5-FU. Therefore, decreased level of TS, makes gastric cancer cell more sensitive to 5-FU17. It is possible, that pancreatic cancer cells ICPD47 interacts with 5-FU in similar way. In different study, Liu et al. determined that 5-FU in combination with another Hsp90 inhibitor, SNX-2112, showed an antagonistic effect on human oesophageal cancer cell viability4. In their opinion, 5-FU could compete with SNX-2112 in binding to Hsp90 N-domain, or induced the cancer signal paths that were inhibited by SNX-2112.In this study, it was determined that different Hsp90 inhibitors in combination with the same anticancer agent exhibiting the same p < 0.05). The ICDPD47 combination with GEM had greater effect on PANC-1 spheroid growth (p < 0.05) than individual compounds. According to scientific literature, pancreatic cancer cell lines are different in genotype and phenotype, so their aggregation qualities and spheroid formation abilities are different. Therefore, it could determine different spheroid growth kinetics between MIA PaCa-2 and PANC-1 cell lines. Overall, the combinations of compounds had greater potency in 2D monolayer cultures than in 3D spheroid cultures. The diffusion of the compounds to inner layers of spheroid could be limited due to high density of the cells and the increased levels of structures of extracellular matrix, which makes a natural barrier similar in solid tumors, therefore only the outer layers could be affected18. Also, the genes related to increased resistance to anticancer agents could be more activated in spheroids than in monolayer cells19.To our knowledge, no studies have been conducted to evaluate combination effects of Hsp90 inhibitors and anticancer agents in pancreatic cancer spheroids. The combination of ICPD47 with 5-FU had a greater effect on MIA PaCa-2 spheroid growth than individual compounds , and DOX hydrochloride and 5-FU were purchased from Sigma-Aldrich Co. .Hsp90 inhibitors ICPD47 and ICPD62 were synthesized at Vilnius University, Lithuania as previously described2 at 37 °C.Human pancreatic cancer cell lines MIA PaCa-2 and PANC-1 were obtained from the American Type Culture Collection . Human foreskin fibroblast cells HF were provided by prof. Santos A. Helder . All cell lines were cultured in Dulbecco’s Medium GlutaMAX (DMEM Glutamax) medium. The medium was supplemented with 10,000 U/mL penicillin, 10 mg/mL streptomycin, and 10% fetal bovine serum. The medium and supplements were purchased from Gibco . Cells were maintained in humidified atmosphere containing 5% COEC50  of the compounds were calculated.Compound effect on cell viability was established using 3--2,5-diphenyltetrazolium bromide  assay. The MIA PaCa-2 and PANC-1 cells were seeded in 96-well plates in a volume of 100 µL . After 24 h preincubation, the cells were treated with 100 µL of different concentrations of Hsp90 inhibitors and anticancer agents. The medium without cells was used as a positive control, and the medium with 0.25% DMSO (Sigma-Aldrich Co.) served as a negative control. After 72 h, the cells were incubated with 20 µL 5 mg/mL MTT solution (Sigma-Aldrich Co.) for 4 h under the same conditions. The absorbance was measured at wavelengths of 570 and 630 nm, and the EC50 of each compound was determined as described previously. To evaluate the effect of combination treatment, different combinations of Hsp90 inhibitors and anticancer drugs in three constant ratios  of EC50 were prepared and serial dilutions were made. The combinations of the compounds were added to the 96-well cell plates with cells after 24 h preincubation and incubated for 72 h. After incubation the MTT assay was performed. To calculate the combination indices (CI), the CompuSyn software was used  that is based of Chou-Talalay method8.The 23. The pancreatic cancer cells were mixed with human fibroblasts in a ratio of 1:1 in order to better represent the tumour microenvironment24. The cells were incubated with nanoparticles NanoShuttle  for 8 h. The cells were resuspended and seeded into ultra-low attachment 96-well plates in a volume of 100 µL . The plate was placed on magnetic drive and incubated in humidified atmosphere containing 5% CO2 at 37 °C for 72 hours until spheroids were formed. The formed spheroids were imaged using light microscopy (Olympus TH4-200). Then, the medium was replaced with the fresh one, containing different concentrations of separate compounds and their combinations in a ratio of 1:5 at the concentrations when fa was 0.5. The photographs of spheroids were taken every 48 h, using Olympus cellSens Standard 1.16 software . The medium was replaced every 96 h25.Spheroids were formed from MIA PaCa-2 and PANC-1 cells using 3D Bioprinting methodThe effect of Hsp90 inhibitors, anticancer agents, and their combinations on spheroid growth was examined by measuring the size change of spheroids using ImageJ software .EC50 values of single Hsp90 inhibitors and the combination of inhibitors and hyperthermia.The cell suspension was prepared and seeded as described previously in cell viability assay in two 96-well plates. After 24-hour preincubation, one plate was heated in a water bath at 43 °C temperature for 30 minutes. Then the cell viability assay (as described previously) was performed to establish p < 0.05 was considered as the level of significance.Statistical analysis was performed using Microsoft Office Excel 2007 software . All experiments were performed in at least triplicate independent measurements and the obtained values were reported as mean ± standard deviation. Student’s t-test was used and p-values were calculated. A value of This article does not contain any studies with human participants or animals performed by any of the authors.For this type of study, formal consent is not required.Supplementary data


The dysfunction of salivary glands commonly induces dry mouth, infections, and dental caries caused by a lack of saliva. This study was performed to determine the genetic and functional changes in salivary glands using a klotho (-/-) mouse model. Here, we confirmed the attenuation of KLF4 expression in the salivary glands of klotho (-/-) mice. Soluble klotho overexpression induced KLF4 transcription and KLF4-mediated signaling pathways, including mTOR, AMPK, and SOD1/2. Silencing klotho via siRNA significantly down-regulated KLF4 expression. Additionally, we monitored the function of salivary glands and soluble klotho and/or KLF4 responses and demonstrated that soluble klotho increased the expression of KLF4 and markers of salivary gland function  in primary cultured salivary gland cells from wild type and klotho (-/-) mice. In a 3D culture system, cell sphere aggregates were observed in soluble klotho- or KLF4-expressing cells and exhibited higher expression levels of salivary gland function-related proteins than those in nontransfected cells. These results suggest that activation of the klotho-mediated KLF4 signaling pathway contributes to potentiating the function of salivary glands. Salivary gland dysfunctional changes occur with reduced salivary flow and dry mouth (xerostomia) and commonly involve oral dysfunction, tooth structure deterioration, and infection through reduced salivation , 2. AnatKlotho is a transmembrane protein and a putative antiaging agent. Klotho-knockout mice demonstrate age-related phenotypes, such as a short lifespan, growth retardation, infertility, skin atrophy, hypoglycemia, hyposalivation, hyperphosphatemia, ectopic calcification, osteoporosis and pulmonary emphysema , 8.2O2-induced apoptosis and senescence [The membrane klotho type acts as a coreceptor of fibroblast growth factor 23 (FGF23) and is capable of regulating phosphate and vitamin D metabolism , 10. FGFnescence . Additionescence , 16.+/K+-ATPase (Atp1a2), Ca2+-ATPase (Atp2a1), epidermal growth factor (EGF), and nerve growth factor (NGF), which have been suggested to regulate submandibular salivary gland function, were significantly decreased. In a network constructed by the differentially expressed genes induced by klotho depletion, proliferator-activated receptor-γ (PPARγ), which regulates energy homeostasis and insulin sensitivity, was located at the core [In our previous study, we analyzed the gene expression profiles of salivary glands from klotho-deficient mice (klotho -/-). Microarray analysis showed that the expression levels of alpha2-Nathe core . RecentlTherefore, this study aimed to evaluate salivary gland functional changes by the soluble klotho/KLF4 signaling pathway, and to provide further baseline information for preclinical research on salivary gland dysfunction related to aging.To determine the tissue level expression of KLF in klotho (-/-) mice, we analyzed KLF4 expression in 8 organs  harvested from four-week-old klotho (-/-) mice. Quantitative RT-PCR revealed that the mRNA levels of KLF4 were decreased to different extents—from 2- to 7-fold—in several organs. The salivary glands, skin, brain, and spleen exhibited significantly decreased KLF4 expression in klotho (-/-) mice compared to that in these organs of wild-type mice . SimilarTo elucidate the relationship between klotho and KLF4 gene expression, we evaluated KLF4 mRNA expression using real-time quantitative RT-PCR in wild-type and klotho (-/-) mouse embryonic fibroblasts (MEFs). Depletion of klotho led to significantly diminished mRNA and protein levels of KLF4 –2C, and To directly assess the functional contribution of soluble klotho to KLF4 signaling, we investigated the expression changes in KLF4-related genes upon the overexpression of soluble klotho in MEFs. We first evaluated the overexpression of soluble klotho in soluble klotho-transfected MEFs by real-time quantitative RT-PCR and observed abundant KLF4 mRNA expression –3D. As sWe next assessed the effect of soluble klotho depletion on KLF4-related protein expression. The inhibition of soluble klotho by siRNA was detected by real-time PCR and Western blot analysis in HEK293 cells, and reduced expression of KLF4 and FOXO1 was observed. KLF4 protein expression was also inhibited in siRNA soluble klotho-transfected cells –4D.To further clarify whether KLF4 depletion modulates soluble klotho-induced KLF4 signaling, we knocked down KLF4 by siRNA in HEK293 cells. As shown in Single-cell suspensions obtained by mechanical and enzymatic dissociation of klotho (-/-) mouse salivary glands were grown in our culture system as described in the Materials and Methods. In klotho (-/-) PSGCs, we examined the expression of KLF4 by real-time quantitative RT-PCR and Western blot. Similar to the results shown in To confirm the function of PSGC populations, we examined the expression of salivary gland functional markers, such as α-amylase (AC secretion product), aquaporin 5 (water channel protein), and ZO-1 (tight junction protein). Quantitative reverse transcription PCR (qRT-PCR) was used to validate the altered expression of these genes. Klotho (-/-) PSGCs showed reduced expression of α-amylase and aquaporin-5 compared to that in klotho (+/+) PSGCs. The tight junction protein ZO-1 was also inhibited in klotho (-/-) PSGCs –5E. In cTo determine whether PSGCs induced by soluble klotho or KLF4 transfection could differentiate into functional activating cells, such as salivary gland ACs, we utilized a 3D culture system. First, we observed the induction of typical mesenchymal stem cell (MSC) surface antigen markers, such as CD44 and CD90, which are known to interact with the extracellular matrix, in soluble klotho- or KLF4-expressing wild-type PSGCs . In addiβ-induced renal fibrosis and metastasis [α and IFNγ. In addition, soluble klotho maintained a low plasma phosphate concentration by inhibiting both renal (NaPi-IIa) and intestinal (NaPi-IIb) phosphate transporters [Soluble klotho has been suggested to function as a humoral factor that targets multiple tissues. A recent study demonstrated that soluble klotho attenuated the generation of reactive oxygen species by insulin and IGF-1 signaling , 19. Soltastasis , 21 and sporters , 23. TheIn a previous study, we performed a transcription factor (TF) activation plate array to profile the TF activities affected by soluble klotho expression. TFs play essential roles in regulating gene expression and act as sensors to monitor cellular changes and convert the signals into altered gene expression. We first found that KLF4 expression was significantly increased after soluble klotho transfection (unpublished data). In this study, KLF4 expression was clearly highly abundant and diverse in the salivary gland, followed by the lung, skin, brain and spleen. The high KLF4 expression in salivary glands is consistent with the immunoblot results. Based on this analysis, KLF4 expression was significantly downregulated in the salivary glands of klotho-deficient mice compared with that in wild-type mice. We confirmed that the expression of KLF4 was decreased in klotho (-/-) MEFs relative to that in wild-type cells. Moreover, using gene transfection and RNA interference techniques, we demonstrated that soluble klotho regulated the expression of KLF4. Interestingly, the KLF4 promoter activity was significantly increased in soluble klotho-overexpressing cells. These results strongly indicate that soluble klotho is involved in the regulation of KLF4 expression.KLF4, an essential regulator of stem cells, was found to be specifically expressed in several normal tissues, especially the salivary gland. KLF4 is downregulated during parotid AC terminal differentiation . We defiCip1/Waf1 by recruiting p53 [A previous study suggested that KLF4 primarily contributes to p53-dependent cell cycle arrest by DNA damage , 26. KLFting p53 , 28. ThuWe recently showed that submandibular salivary glands in klotho-deficient mice show a loss of granular ducts and mucous acini compared to those of wild-type mice . Loss ofin vitro characteristics of PSGCs isolated from klotho wild-type and klotho (-/-) mice. The PSGCs of klotho (-/-) mice showed low KLF4 expression compared to those of wild-type mice and inhibited the expression of salivary gland functional markers, such as α-amylase, aquaporin 5, and ZO-1. However, overexpression of soluble klotho induced KLF4 and salivary gland functional markers in PSGCs. In addition, small colonies of epithelial-shaped cells were observed on the culture substrate within 5 days under soluble klotho or KLF4 transfection conditions. These spherical cells were positively labeled with AQP-5 and α-amylase. Notably, strong staining of ZO-1 was observed. The expression of salivary gland functional markers in soluble klotho- or KLF4-overexpressing ACs is shown in Therefore, we examined the critical role of soluble klotho/KLF4 in salivary gland dysfunction. In this study, we investigated the in vitro will express CD44 and CD90 and retain these morphological and phenotypical characteristics after 3D culture. These results showed that soluble klotho-induced cells retain their ability to self-renew as stem cells and may restore the function of the salivary gland.In addition, we demonstrated the expression of stem cell markers (CD44 and CD90) in soluble klotho-induced PSGCs. CD44, a stem cell marker, has previously been reported in serous ACs of human salivary gland tissue , 30. FurIn summary, soluble klotho directly induces cyclin D1/B1 and AMPK and regulates p53/p21 and SOD1/2 signaling by inducing KLF4. Our findings suggest a critical role for soluble klotho/KLF4 in the functional restoration of the salivary gland and selectively inducing the proteins necessary for salivary gland function. Soluble klotho/KLF4 signaling in PSGCs induced the expression of salivary gland functional markers . Additionally, 3D culture analysis showed that spherical cells were formed in soluble klotho- or KLF4-induced PSGCs. We predict that the expression of the progenitor cell markers CD44 and CD90 in soluble klotho-induced PSGCs may at least partially explain their regenerative potential. We expect that our results will contribute to better understanding soluble klotho signaling in the salivary gland and the development of potential cell therapies for patients with irreversible salivary gland function loss.2/95% air atmosphere at 37°C. Immortalized human salivary gland ACs were cultured on keratinocyte serum-free medium  containing 100 U/ml penicillin and 100 μg/ml streptomycin.MEFs were derived and cultured from SAMP1/kl+/+ and SAMP1/kl-/- 13-day-old embryos that were obtained by mating SAMP1/kl+/- mice with SAMP1/kl+/- mice  and main2 (6.25 mM) at 37°C for 40 min. Dissociated cells were centrifuged at 1000 rpm for 5 min, and the cell pellet was resuspended in the culture medium. The cells were plated in a 12-well plate at a density of 5 × 104 cells/well in DMEM/F-12 (Gibco) medium supplemented with 100 U/mL penicillin G, 100 μg/ml streptomycin, 20 ng/mL EGF (Sigma-Aldrich), 20 ng/mL basic fibroblast growth factor (Gibco), 1/100 N2 supplement (Gibco), 10 μg of insulin-transferrin-selenium (Gibco), and 1 μM dexamethasone (Sigma-Aldrich) and incubated in 5% CO2 at 37°C. Fresh medium was added every three days. After 4-5 passages, the expression of salivary gland functional markers, such as α-amylase (AC secretion product), aquaporin 5 (water channel protein), and ZO-1 (tight junction protein), was examined.Submandibular gland tissues were harvested from 4- or 8-week-old wild-type and klotho (-/-) mice. The tissues were dissociated from cervical fascia and connective tissues under a dissecting microscope and gently collected in phosphate-buffered saline (PBS). Freshly dissociated tissues were washed twice with DMEM and Ham’s F-12 medium  supplemented with 100 U/mL penicillin G and 100 μg/ml streptomycin (Gibco), minced with scissors, and incubated with DMEM/F12 medium containing collagenase type II (0.025%), hyaluronidase (0.04%) and CaCl(target gene) − CT  and ΔΔCt = ΔCt  − ΔCt (control group). The relative expression levels of related genes were determined by the 2−ΔΔCT method, and the experiments were repeated three times.Total RNA was extracted with TRIzol reagent (Invitrogen). Reverse transcription was performed on 1 μg of total RNA using oligo dT primers and M-MLV Reverse Transcriptase (Invitrogen) in a final volume of 20 μl for 5 min at 65°C followed by 1 h at 37°C. Real-time quantitative PCR was performed using the SYBR Green kit  according to the manufacturer’s instructions. The expression levels of soluble klotho, KLF4, and FOXO1 mRNA were normalized to those of GAPDH for each sample. The following primers were used for real-time quantitative PCR detection: soluble klotho, 5′-GCTCTGCTTCCGCC ACTT-3′ (forward) and 5′-GCATGAGCCAGGAGGAG G-3′ (reverse); KLF4, 5′-TTCCAACTCGCTAACCCAC C-3′ (forward) and 5′-GCTCGGGACTCAGTGTAG-3′ (reverse); FOXO1, 5′-AGACCCGGGTTCTTTGACAC-3′ (forward) and 5′-TTGTCATAGGCTTCCCACCA-3′ (reverse); amylase, 5′-GGTGCAACAATGTTGGTGTC-3′ (forward) and 5′-ACTGCTTTGTCCAGCTTGAG-3′ (reverse); Aqua5, 5′-CGACCGTGTGGCTGTGGTCA-3′ (forward) and 5′-GTGCCGGTCAGTGTGCCGTC-3′ (reverse); ZO-1, 5′-CGAGGCATCATCCCAAATAAGA AC-3′ (forward) and 5′-TCCAGAAGTCTGCCCGATC AC-3′ (reverse). The samples were quantified based on the threshold cycle by interpolation from the standard curve. The reliability of the PCR results was evaluated by a solubility curve as follows: ΔCt = CT KLF4 siRNA was obtained in the form of Silencer Select Validated siRNA (Applied Biosystems). The sense sequence of the KLF4 siRNA was 5′-CUCGUUCUCG GCUUGUCUA (dTdT)-3′, and the antisense sequence was 5′-UAGACAAGCCGAGAACGAG (dTdT)-3′. The cells were transfected with siRNA (20 nM) using Lipofectamine 2000 siRNA Transfection Reagent  according to the manufacturer’s instructions. The cells were harvested 48 h after transfection. Total cell lysates were separated by SDS-PAGE and analyzed by Western blot as described above.Cells were lysed, and proteins were extracted in a radio immunoprecipitation assay buffer  and complete protease inhibitor cocktail (Sigma-Aldrich). The lysates were obtained after centrifugation, and the protein concentration was measured using the BCA protein assay kit . The lysates were separated by 12% SDS-PAGE. After transfer to PVDF membranes , the membranes were blocked for 1 h at room temperature with 5% nonfat dry milk in TBS-T buffer. The blocked membranes were then incubated with primary antibodies (1:1000 dilution) against klotho (10E2), KLF4 (3F3), FOXO1 (7G6C5), phospho-mTOR (D15C3), phospho-AMPK (D2E5), and p21 (D1F2), which were purchased from Cell Signaling Technology. The cyclin D1, cyclin B1, SOD1, SOD2, p70S6K, and actin antibodies were purchased from Santa Cruz Biotechnology . The membranes were washed 3 times with TBS-T buffer and then incubated with secondary antibodies conjugated to HRP  for 1 h at room temperature. The immunoreactive proteins were visualized with ECL detection reagents (Thermo).The pGL4-KLF4 promoter (-2.046 bp from the start codon) was generously provided by Dr. Yoshida . HEK293 cells were transfected with 1 μg of the pGL4-KLF4 Luc plasmid and 1 μg of Tk-Renilla by Fugene 6 HD (Promega) reagent and harvested after 48 h. Additionally, the cells were transfected with pcDNA3-sKL and/or pcDNA3-KLF4 using Fugene 6 HD reagent for 48 h. Transfected cells were lysed and measured with the Dual-Luciferase Reporter Assay System  according to the manufacturer’s instructions. Relative luciferase units were normalized by the Tk-Renilla luciferase value in each sample. Each experiment was performed in triplicate and repeated twice.4 cells/well were seeded on Matrigel, and 3D spheroids were grown at 37°C in 5% CO2. For the generation of single-cell suspensions from 3D spheroids, spheres were isolated by removing media and adding 0.5 ml of Dispase (1 mg/ml) directly to the Matrigel followed by incubation for 1 h at 37°C in 5% CO2 to digest the Matrigel. The spheres were washed in PBS, centrifuged at 1200 rpm for 5 min, dissociated with TrypLE Express (Gibco) and passed through a 40 μm filter to remove clumps and obtain single-cell suspensions. The cells were then plated and grown as monolayers in either serum-free medium (SFM) or serum-containing medium (SCM).One hundred microliters of Matrigel  in 48-well chambers was allowed to solidify in a 37°C incubator for 1 h. Upon solidification, 5 x 10+ confocal microscope (Japan). The average intensity value of the nonspecifically bound secondary antibody was subtracted from all images using the ImageJ program.The presence of aquaporin-5 and ZO-1 in 3D cultured cells was confirmed by indirect immunofluorescence staining as described previously . BrieflyP-values <0.05 were considered statistically significant.Data are presented as the mean ± SEM. Statistical significance between groups was determined by Student’s t test and one-way analysis of variance (ANOVA). Supplementary Figures


While some studies confirmed a (near)‐essential role for BUB1 in the SAC . To this end, we performed live cell imaging of cells treated with a high dose of nocodazole to trigger a full SAC response. We observed that both ∆BUB1 clones were able to establish a functional SAC as cells displayed a prominent arrest in response to nocodazole treatment, not significantly different from WT HAP1 cells or our previously published BUB1 KO cells (∆BUB1 “Ex3”), that were generated by the stable integration of a Blasticidin resistance cassette in exon 3  have been unsuccessful. Although generating knockouts in these cell lines can be technically more challenging due to the presence of multiple alleles, this result could indicate that the loss of BUB1 is not well tolerated in all cell types. This possibly reflects a difference in the relative contribution of BUB1 to the SAC between cell lines or a difference in the tolerance toward mild chromosome segregation errors. Thus, although the most reliable way to fully resolve the exact contribution of BUB1 to the SAC in different genetic backgrounds would be to stably eradicate the full gene, this is only feasible if this is a tolerated condition.AppendixClick here for additional data file.Review Process FileClick here for additional data file.


There are errors in the Abstract. The correct Abstract is as follows: Anthropogenic infrastructure can negatively affect wildlife through direct mortality and/or displacement behaviors. Some tetranoids (grouse spp.) species are particularly vulnerable to tall anthropogenic structures because they evolved in ecosystems void of vertical structures. In western North America, electric power transmission and distribution lines (power lines) occur in sagebrush (Artemisia spp.) landscapes within the range of the greater sage-grouse . The U.S. Fish and Wildlife Service recommended using buffer zones near leks to mitigate the potential impacts of power lines on sage-grouse. However, recommended buffer distances are inconsistent across state and federal agencies because data are lacking. To address this, we evaluated the effects of power lines on sage-grouse breeding ecology within Utah, portions of southeastern Idaho, and southwestern Wyoming from 1998–2013. Overall, power lines negatively affected lek trends up to a distance of 2.3 km. Power lines did not affect lek persistence. Female sage-grouse avoided transmission lines during the nesting and brooding seasons at distances up to 1.1 and 1.2 km, respectively. Nest and brood success were negatively affected by transmission lines, however no maximum effect distance was identified. Distribution lines did not appear to affect sage-grouse habitat selection or reproductive fitness. Our analyses demonstrated the value of sagebrush cover in mitigating potential power line impacts. Managers can minimize the effects of new transmission power lines by placing them in existing anthropogenic corridors and/or incorporating buffers at least 2.3 km from active leks. Given the uncertainty we observed in our analyses regarding sage-grouse response to distribution lines coupled with their role in providing electric power service directly to individual consumers, we recommend that buffers for these power lines be considered on a case-by-case basis. Micrositing to avoid important habitats and habitat reclamation may reduce the potential impacts of new power line construction.


Cytotoxic activities of the above mentioned compounds against five human carcinoma cell lines, namely the human nasopharyngeal epidermoid carcinoma cell line (KB), human cervical carcinoma cell line (CasKi), human colon carcinoma cell line (HCT 116), human hormone-dependent breast carcinoma cell line (MCF7) and human lung carcinoma cell line (A549); and non-cancer human fibroblast cell line (MRC-5) were investigated. Compound 5 possessed very remarkable cytotoxic activity against KB cells, with an IC50 value of 0.81µg/mL. This is the first report on the cytotoxic activities of the compounds isolated from Pereskia bleo. Dihydroactinidiolide ( Pereskia bleo (Kunth) DC. (Cactaceae) are used traditionally in Malaysia for the treatment of cancer, high blood pressure, diabetes and diseases associated with rheumatism and inflammation. They are also used as remedy for the relief of gastric pain, ulcers and for revitalizing the body  and cytotoxic investigation on all isolated compounds against five human carcinoma cell lines, namely the human nasopharyngeal epidermoid carcinoma cell line (KB), human cervical carcinoma cell line (CasKi), human colon carcinoma cell line (HCT 116), hormone-dependent breast carcinoma cell line (MCF7) and human lung carcinoma cell line (A549) and non-cancer human fibroblast cell line (MRC-5).In the present study, we report further progress in ongoing research on 4), 2,4-di-tert-butylphenol (5), α-tocopherol (6) and phytol (7) were obtained from Pereskia bleo as previously described by Sri Nurestri et al.  + (15), 137 (8), 111 (100), 109, 67. Compound 1 was identified by comparison of its mass spectral data with NIST mass-spectral library  with reported data . In contrast, the cytotoxic effect of the crude methanol extract on the MCF7 cell line gave IC50 of 39.0 µg/mL (mild) whilst two isolated compounds 2,4-di-tert-butylphenol (5) and α-tocopherol (6), showed good inhibitory activities with IC50 values of 5.75 and 7.5 µg/mL, respectively.However, the observed activity might be due to synergism between compounds present in the plant extract. The synergism among these compounds which contribute to the cytotoxic activity, is not only dependent on the concentration of the compounds, but also on the structure and interaction(s) between the compounds . This ca5) displayed very remarkable cytotoxic activity against KB cells with an IC50 value of 0.81 µg/mL and strong cytotoxicity against MCF7 (IC50 5.75 µg/mL), A549 (IC50 6 µg/mL) and CasKi cells (IC50 4.5 µg/mL). This in vitro data of 2,4-di-tert-butylphenol (5) support the findings that phenolic antioxidants exert cytoctoxic activity against cancer cells  did not display cytotoxic effects against the tested cell lines. The results obtained here were in agreement with published data  and finally MeOH (7.6 L). The CHCl3 fraction was concentrated to give a dark brown residue (3.47 g). The brown residue was subjected to a Si-gel CC initially eluting with a gradient of hexane followed by hexane enriched with increasing percentages of CH2Cl2, monitoring with TLC. The volume of each fraction was 25 mL. The mixture of sterols (20.5 mg) was obtained from the fraction upon elution with CH2Cl2-hexane (3.5: 6.5). Further elution with CH2Cl2 yielded a mixture (206.7 mg) containing 1. Purification of 1 was obtained through preparative-TLC using CHCl3 as the developing solvent to yield pure compound 1 (5.4 mg). β-Sitosterol  kept at 37°C in a humidified atmosphere. Human nasopharyngeal epidermoid carcinoma cell line (KB), human cervical carcinoma cell line (CasKi), human colon carcinoma cell line (HCT 116), human hormone-dependent breast carcinoma cell line (MCF7), human lung carcinoma cell line (A549) and non-cancer human fibroblast cell line (MRC-5) were purchased from the American Tissue Culture Collection . KB cells were maintained in Medium 199 (Sigma), CasKi, A549 and MCF7 cells in RPMI 1640 medium (Sigma), HCT 116 in McCOY’S 5A Medium (Sigma) and MRC-5 cells in EMEM  (Sigma), supplemented with 10% foetal bovine serum , 100 µg/mL penicillin or streptomycin  and 50 µg/mL of fungizone . The cells were cultured in a 5% CO4/well) were seeded in 96-well microtiter plates (Nunc) and allowed to grow for 24 hours before treatment. After 24 hours of incubation, the cells were treated with six different concentrations (0.1-100 µg/mL) of test compounds, in three replicates. The plates were further incubated for 72 h at 37°C in a 5% CO2 incubator. A stock solution was initially obtained by dissolving the test compounds in DMSO. Further dilution to different tested concentrations were then carried out ensuring that the final concentration of DMSO in the test and control wells was not in excess of 1% (v/v). No effect due to the DMSO was observed. Doxorubicin was used as the positive control. The well containing untreated cells was the negative control. At the end of the incubation period, the media were replaced with medium containing 50 µg/mL of Neutral Red. The plates were incubated for another 3 hours to allow for uptake of the vital dye into the lysosomes of viable and injured cells. After the incubation period, the media were removed and cells were washed with the neutral red washing solution. The dye was eluted from the cells by adding 200 µL of Neutral Red resorb solution and incubated for 30 minutes at room temperature with rapid agitation on a microtiter plate shaker. Dye absorbance was measured at 540 nm using a spectrophotometer ELISA plate reader. The average data from triplicates were expressed in terms of killing percentage relative to negative control. The percentage of inhibition (%) of each of the test samples was calculated according to the following formula:The Neutral Red cytotoxicity assay is based on the initial protocol described by Borenfreund and Puerner  with som50 value. The IC50 value is the concentration of test compounds that cause 50 % inhibition or cell death, averaged from the three experiments, and was obtained by plotting the percentage inhibition versus concentration of test compounds. According to US NCI plant screening program, a plant extract is generally considered to have active cytotoxic effect if the IC50 value, following incubation between 48 to 72 hours, is 20 μg/mL or less, while it is 4 μg/mL or less for pure compounds [50 value corresponds to a significant or non-significant cytotoxicity depends on the sensitivity of the cell line. Cytotoxicity of each sample is expressed as ICompounds ,13,22,23Pereskia bleo [1, 5, 6 and 7. Although the cytotoxicity of these compounds and mixture are not as effective as doxorubicin, in comparison to the latter they have low toxicity against normal MRC5 cell line. The cytotoxicity assay used in the present study could only provide important preliminary data to help select plant extracts or isolated compounds with potential antineoplastic properties for future work. A detailed investigation on the mechanism of cell death would provide more convincing evidence. An investigation into this phenomenon is now underway and will be reported in due course. The resulting information will certainly contribute to a better understanding of the anti-carcinogenic activity of the natural constituents in Pereskia bleo. In conclusion, and depending on the cell lines used, the cytotoxic activities observed for kia bleo  are ascrPereskia bleo has been traditionally used for the treatment of cancer and the findings of the current study thus provide scientific validation on the use of the leaves of Pereskia bleo. In view of the increasing popular consumption of medicinal plants as alternative therapy, it is therefore necessary to conduct serious research to support the therapeutic claims and also to ensure that the plants are indeed safe for human consumption.


Sarcomas are rare mesenchymal malignant tumors with unique biological and clinical features. Given their diversity, heterogeneity, complexity, and rarity, the clinical management of sarcomas is quite challenging. Cell lines have been used as indispensable tools for both basic research and pre-clinical studies. However, empirically, sarcoma cell lines are not readily available. To understand the present status of sarcoma cell lines and identify their current challenges, we systematically reviewed reports on sarcoma cell lines. We searched the cell line database, Cellosaurus, and categorized the sarcoma cell lines according to the WHO classification. We identified the number and availability of sarcoma cell lines with a specific histology. We found 844 sarcoma cell lines in the Cellosaurus database, and 819 of them were named according to the WHO classification. Among the 819 cell lines, 36 multiple and nine single cell lines are available for histology. No cell lines were reported for 133 of the histological subtypes. Among the 844 cell lines, 148 are currently available in public cell banks, with 692 already published. We conclude that there needs to be a larger number of cell lines, with various histological subtypes, to better benefit sarcoma research.  Sarcomas are rare mesenchymal malignant tumors with unique biological and clinical features. Sarcomas are unique malignancies for several reasons. First, sarcomas originate from diverse mesenchymal tissue lineages such as adipose, muscle, fibrous, cartilage, nervous, and vascular tissues, or bone. Since these tissues are distributed throughout the human body, sarcomas can occur in almost all organs. Second, sarcomas are heterogeneous diseases that are pathologically grouped into more than 70 described subtypes . The hisRecent advances in genomic technology using next-generation sequencing have enabled the classification of sarcomas, which did not fit into known specific diagnostic categories . Such clGiven their diversity, heterogeneity, complexity, and rarity, the clinical management of sarcomas is quite challenging. However, there are many successful examples of innovative drugs for sarcomas. For example, a tyrosine kinase inhibitor, imatinib mesylate, was originally developed for the treatment of chronic myelocytic leukemia, which has a unique chromosome translocation; later, imatinib mesylate was repurposed to gastrointestinal stromal tumor (GIST), which is characterized by mutations or overexpression of c-kit and PDGFR ,16,17,18Other drugs include trabectedin, pazopanib, eribulin, olaratumab, and denosumab. Trabectedin, which binds to the minor groove of DNA to cause DNA damage, demonstrated evidence of cytotoxic activity against soft tissue sarcomas ,25,26,27This recent progress in the development of novel anti-cancer drugs suggests improvement in the clinical outcomes of patients with sarcomas in the near future ,47,48. HCell lines have been used as indispensable tools for both basic research and pre-clinical studies. Since animal cell culture became a common laboratory technique in the mid-1900s, it has served as a model for human cancer research. Since cell lines are maintained in artificial tissue culture conditions, there are critical arguments about the utility of cell lines; however, the advantages of other cancer models, such as xenografts  and orgaTo date, different sarcoma cell lines have been developed. These cell lines represent a useful experimental model to examine the hypothesis about the etiology of diseases, to evaluate the molecular mechanisms of cancer progression, and to examine the effect of potent anti-cancer drugs at the cellular and subcellular levels. At the same time, besides the obvious utilities of cell lines in cancer research, researchers may be empirically aware that sarcoma cell lines are not readily available, probably due to the rarity of the disease, and a lack of proper cell lines hinders basic studies and development of effective therapies for sarcomas. In this review, we provide an overview of the current status of reported sarcoma cell lines, and finally discuss what types of sarcoma cell lines need to be established, what system needs to be created to promote sarcoma research using cell lines, and what biological studies need to be performed to improve the present status of sarcoma cell lines.https://web.expasy.org/cellosaurus/) and searched using ontology: human cell lines were searched with the term ‘NCBI_TaxID=9606; ‘Homo sapiens’ in ‘Species of origin’, and sarcoma cell lines were searched with a term that was one of the children of the term ‘NCIt:C3810 Connective and Soft Tissue Neoplasm’ in ‘Disease’. The NCI thesaurus ontology file was downloaded from the website of NCI Thesaurus (https://ncit.nci.nih.gov).All potentially relevant cell lines were identified by searching the Cellosaurus database  ,57. The The following criteria had to be met for a cell line to be included in this review: cell line established from human patients with connective and soft tissue neoplasm, regardless of histology or original sites. Cell lines derived from other cell lines and modified by genes or reagents were considered as duplicates and excluded from the data analysis.https://www.python.org/): cell line name, disease, publications, and cell line collections.The following data were examined for each cell line using Python version 3.6.1  for published sarcoma cell lines. The publications in PubMed were also considered for investigation because these cell lines are available from public cell banks or published in academic journals and were, therefore, somehow assured by the public organizations or research community. In addition, the cell lines and their relevant data were supposed to be easily obtainable.In the Cellosaurus database, the availability of cell lines was examined from the following cell banks: AddexBio, ATCC, BCRC, BCRJ, BEI_Resources, CBA, CCLV, Cell_Biolabs, CLS, Coriell, DGRC, DiscoverX, DSMZ, ECACC, FCDI, ICLC, Imanis, IZSLER, JCRB, KCB, KCLB, KYinno, Millipore, MMRRC, NCBI_Iran, NCI-DTP, NHCDR, NIH-ARP, NISES, RCB (Riken), RSCB, TCB, TKG, TNGB, and WiCell. In addition, we searched the Ximbio website . Those modified cell lines are most common in osteosarcoma (275 cell lines). Among the 275 cell lines, U2OS cell lines are most commonly used as original cell lines for modifications. We found that there are cell lines for which the reported histology did not match that in the WHO classification (Table 3).Among the 819 cell lines originally derived from patients with connective and soft tissue neoplasm, 139 cell lines were available from the public cell banks , while 6Among the 819 cell lines originally derived from patients with connective and soft tissue neoplasm, 674 cell lines were cited in the PubMed database , while 1The cell lines whose establishment was reported in academic journals that are cited in the PubMed database can be useful research resources because the relevant data of cell lines are available from the published papers. Among the 844 original cell lines, there were 692 cell lines that have been published: among them, 108 cell lines are available from public cell banks A Supple. Among tA lack of sarcoma cell lines is empirically noticed in the research community; it is important to know their availability from a practical view point. In this review, we investigated the current status of sarcoma cell lines to reveal what cell lines have to be established to promote sarcoma research. The cell line database, Cellosaurus, used in this study includes more than one hundred thousand cell lines and is frequently updated. Thus, Cellosaurus is an adequate cell line database for investigation.We grouped the cell lines according to the histology of their original tumor. We found that 45 histological subtypes were covered by the currently reported cell lines, while 133 were not. Considering the diversity and complexity of sarcomas, we need more cell lines that represent the different histological subtypes. In addition, we found that multiple cell lines were established for 36 histological subtypes, with a single cell line reported for nine subtypes. During the course of the cell line establishment, clonal selection and expansion may occur, and only limited cell populations may survive under tissue culture conditions. To understand the sustainability of the original characteristics of the established cell lines, the capability of tumor tissue formation and the histology of the formed tumors can be evaluated by xenograft experiments. In addition, patient-to-patient variations are clinically considerable even if they have tumors with the same histology. Therefore, no single cell line can represent the characteristics of whole tumor tissues; we need to use multiple cell lines. In this sense, we also need more cell lines for sarcomas that already have corresponding cell lines.Cell lines are most frequently established from Ewing’s sarcoma and osteosarcoma samples. However, the absolute number of patients with Ewing’s sarcoma and osteosarcoma is small according to medical statistics; undifferentiated pleomorphic sarcoma, liposarcoma, and leiomyosarcoma are more common sarcomas . Thus, tClassification of Tumours of Soft Tissue and Bone [The histological diagnosis of original tumors of cell lines may need to be updated in cases where the name of cell lines did not match the official classification. Among the 844 sarcoma cell lines investigated, 42 were not named according to the 2013 World Health Organization and Bone . The diaUnfortunately, cell lines are not always deposited in cell banks. We found that only 139 of 819 sarcoma cell lines named according to the WHO classification were deposited in public cell banks. Probably, the rest of the cell lines can be provided upon request by researchers. The current cell bank systems may rely on researchers and institutes to undertake the cell line establishment. Establishing novel cell lines costs a considerable amount of resources, such as time and money; furthermore, because cell lines are properties of the institutes to which researchers are affiliated, it may be difficult to deposit all cell lines in public cell banks and share them with other researches. As the establishment of cell lines itself is not necessarily a novel discovery, nor would the publication be in high-impact journals, researchers may not be motivated to establish and share cell lines. A system to motivate cell line establishers and their institutes may be required to improve the availability by depositing cell lines.This systematic review has several limitations. First, although the genetic background and biological characteristics of some but not all cell lines were reported in publications, this review did not summarize those data. In our research, 692 cell lines were reported in previous papers, and 108 of them were deposited in cell banks . AlthougCell lines have been considered a valuable tool for both basic research and pre-clinical studies. The functional significance of genetic products such as mRNA, miRNA, and proteins can be clarified using living cells, and cell lines are an indispensable research resource. In the preclinical evaluation of new drugs, their tumor suppressive effects and mode-of-action are also investigated using cell lines. Although the predictive power of cell lines can be undermined by the selective pressures during the process of establishment and long-term passaging, a great advantage of cell lines is that the examinations can be done in a high-throughput manner with relatively low costs. Patient-derived xenografts (PDXs) may complement the inherent drawbacks of cell lines, because PDXs may retain the microenvironmental conditions of the original tumors. However, the manipulation of PDX requires time-consuming and tremendous efforts, and their unstable molecular backgrounds have been revealed at the genome level. Moreover, because the human stromal components in PDX tumors are replaced with mouse ones after several passes, consistent results may be limited in experiments using PDXs. Taken together, cell lines have a unique utility, and they are indispensable in cancer research.We conclude that (1) more sarcoma cell lines representing the various histological types as well as those established by single cell lines are needed to effectively capture the diversity and complexity of the disease; (2) a system is needed to reward the efforts of researchers who establish and deposit cell lines into public cell banks to promote cell line sharing in the research community, and (3) further investigations are required to determine the critical factors determining the success rate of the cell line establishment and create effective experimental protocols.


Cisplatin is one of the major drugs used in oral cancer treatments, but its usage can be limited by acquired drug resistance. In this study, we established three cisplatin-resistant oral squamous cell carcinoma (OSCC) cell lines and characterized them using cell viability assays, qPCR, Western blotting, FACS, immunofluorescence, and wound healing assays. Three OSCC cell lines  underwent long-term exposure to cisplatin, eventually acquiring resistance to the drug, which was confirmed by an MTT assay. In these three newly established cell lines , overexpression of multidrug resistance (MDR)-related genes was detected by qPCR and Western blotting. The cell lines displayed an increase in the functional activities of breast cancer resistance protein (BCRP) and multidrug resistance protein1 (MDR1) by rhodamine 123 and bodipy FL prazosin accumulation assays. Moreover, the cisplatin-resistant cells underwent morphological changes, from round to spindle-shaped, increased expression of epithelial-to-mesenchymal transition (EMT)-related molecules such as N-cadherin, and showed increased cell migration when compared with the parental cell lines. These results suggest that these newly established cell lines have acquired drug resistance and EMT induction.  Oral cancer is an increasingly global disease ,2, howevCisplatin, a platinum-based drug, is one of the standard first-line chemotherapeutic agents used to treat OSCC . UnfortuEpithelial-to-mesenchymal transition (EMT) is a process by which epithelial cells lose their cell polarity and cell–cell adhesion, and gain mobility and invasiveness to become mesenchymal stem cells . In mostThe purpose of our study was to establish three cisplatin-resistant human OSCC cell lines, and evaluate their biological characteristics compared to their respective parental cell lines.Three OSCC cell lines, YD-8, YD-9, and YD-38, were continuously exposed to stepwise increasing concentrations of cisplatin over a one-year period . An MTT assay was performed to determine whether the cell lines had developed cisplatin-resistance. MDR1 (>2.4-fold change), BCRP (>7.2-fold change) and MRP3 (>2.5-fold change) were upregulated. In YD-9/CIS cells, MDR1 (>7.5-fold change), MRP4 (>2.7-fold change), and BCRP (>1.6-fold change) were upregulated. In YD-38/CIS cells, BCRP (>3.7-fold change) was upregulated. We confirmed the expression changes in BCRP and MDR1 by Western blot  showed resistance to paclitaxel. In addition, YD-8/CIS, YD-9/CIS, and YD-38/CIS showed low, medium, and no resistance to CKD-602, respectively. None of the cell lines acquired a resistance to 5-fluorouracil. Therefore, our results show that the three cisplatin-resistant cell lines acquired resistance to cisplatin, and the cross-resistance to different chemotherapeutic drugs was cell-specific.BCRP expression was significantly upregulated in YD-8/CIS and YD-38/CIS cells, and MDR1 expression was significantly upregulated in YD-9/CIS cells; these results were confirmed via Western blotting. Therefore, it has been confirmed that long-term exposure of OSCC cell lines to cisplatin increased the expression of the ABC transporter genes, MDR1 and BCRP. MDR1 is a 170 kDa ATP-dependent membrane transporter that is widely distributed throughout the body and pumps many foreign substances out of cells [One of the common mechanisms of drug resistance is through a reduction in their intracellular accumulation, which is associated with overexpression of ABC transporters ,24. Thusof cells . BCRP isof cells .Next, we compared the functional activities of MDR1 and BCRP between the parental and cisplatin-resistant cells using rhodamine 123 and bodipy FL prazosin accumulation assays. Rhodamine 123, a cationic fluorescent dye, is a good MDR1 substrate. Bodipy FL prazosin, a fluorescent derivative of the quinazoline based α-adrenergic antagonist prazosin, is known as a BCRP substrate ,26,27,28Several studies have shown that MDR is associated with EMT ,31. ThusCisplatin (PubChem CID: 84691) was purchased from Choongwae Pharm. C. (Korea) and CKD-602 (PubChem CID: 123773277) was kindly provided by Chongkundang Pharm. Co. . These drugs were dissolved in distilled water at 1 mg/mL. 5-Fluorouracil (PubChem CID: 3385) was obtained from Sigma  and dissolved in DMSO at 20 mg/mL. Paclitaxel (PubChem CID: 36314) was purchased from Hanmi Pharm. Co. . Each aliquot of the stock solutions was stored at −20 °C until use.2. When culturing the resistant cell lines, the final concentration of cisplatin was maintained at 2 μg/mL. Parental OSCC cell lines  were obtained from the Korean Cell Line Bank , and cisplatin-resistant cell lines  were generated in our lab by exposing the original cell lines to increasing concentrations of cisplatin over a period of one year. The cell lines were maintained in RPMI-1640 medium  supplemented with 10% heat-inactivated FBS  and 1% penicillin-streptomycin  at 37 °C in a humidified atmosphere containing 5% CO4 cells per well in 96-well plates and then treated with cisplatin, 5-fluorouracil, paclitaxel, or CKD-602. Cells were incubated with the drugs for 72 h, after which 0.5 mg/mL of MTT reagent  was added to each well. After 2 h, the medium was discarded, the resulting formazan was dissolved in DMSO, and the absorbance at 570 nm was measured using a SpectraMax Plus 384 microplate reader . For cell viability assays, single cell suspensions were seeded at 1 × 10MDR1, MRP1-6, MRP9, and BCRP was analyzed by a qPCR ViiA7 Real-Time PCR System using PowerUp SYBR Green Master Mix . The following primers were used: MDR1, 5′-CACCCGACTTACAGATGATG-3′ (forward) and 5′- GTTGCCATTGACTGAAAGAA-3′ (reverse); MRP1, 5′-GCCGAAGGAGAGATCATC-3′ (forward) and 5′-AACCCGAAAACAAAACAGG-3′ (reverse); MRP2, 5′-CGACCCTTTCAACAACTACTC-3′ (forward) and 5′-CACCAGCCTCTGTCACTTC-3′ (reverse); MRP3, 5′-GTGGGGATCAGACAGAGAT-3′ (forward) and 5′-TATCGGCATCACTGTAAACA-3′ (reverse); MRP4, 5′-GCTCAGGTTGCCTATGTGCT-3′ (forward) and 5′- CGGTTACATTTCCTCCTCCA-3′ (reverse); MRP5, 5′-CAGCCAGTCCTCACATCA-3′ (forward) and 5′-GAAGCCCTCTTGTCTTTTTT-3′ (reverse); MRP6, 5′-AGGAGGCCCGAGCTTAGAC-3′ (forward) and 5′-CCTGCCGTATTGGATGCTGT-3′ (reverse); MRP9, 5′-ATGCGGTTGTCACTGAAG-3′ (forward) and 5′-GTTGCCTCATCCATAATAAGAAT-3′ (reverse); BCRP, 5′-TGACGGTGAGAGAAAACTTAC-3′ (forward) and 5′-TGCCACTTTATCCAGACCT-3′ (reverse); and GAPDH, 5′-AATCCCATCACCATCTTCCA-3′ (forward) and 5′-TGGACTCCACGACGTACTCA-3′ (reverse) [−ΔΔCT method [Total RNA was extracted from the harvested culture cells in 1.5 mL tubes using an RNA-spin Total RNA Extraction Kit . cDNA synthesis was performed using High-Capacity cDNA Reverse Transcription Kits . The expression of reverse) . The relT method . To investigate the expression levels of multidrug resistant- or EMT-related proteins in the parent and resistant cell lines, we performed a Western blot assay. Whole cell lysates (20 μg) were resolved by 10% SDS-PAGE, and proteins were transferred onto a PVDF membrane . The membrane was incubated overnight at 4 °C with the following antibodies: anti-MDR1 from Abcam , anti-BCRP from Santa Cruz Biotechnology , anti-E-cadherin and -N-cadherin purchased from CellSignaling Technology , and anti-β-actin obtained from Sigma . The immunobands were detected using a ChemiDoc Touch imaging system .5 cells per well in a 6-well plate. The next day, cells were treated with rhodamine 123 (1 μg/mL) or prazosin (250 nM) for 30 min. After incubation, cells were washed twice in ice-cold PBS, harvested by centrifugation at 1000 rpm for 5 min, and resuspended in 500 μL of PBS. Accumulation of rhodamine 123 or prazosin in cells was measured using a FACSCalibur flow cytometer . At least 10,000 events were analyzed for each tube.For the rhodamine 123 and bodipy FL prazosin accumulation assays, cells were seeded at 2 × 10E-cadherin and N-cadherin were analyzed by immunofluorescence in parental and cisplatin-resistant cells. Cells were seeded on cover slips in 6-well plates and incubated overnight at 37 °C. The next day, the cells were washed three times with PBS, fixed with 4% paraformaldehyde for 15 min and permeabilized with 0.1% NP-40 in PBS for 10 min at room temperature. The cells were blocked with 10% FBS in PBS for 30 min and incubated with either anti-E-cadherin or N-cadherin primary antibody (1:50 dilution) overnight at 4 °C. After this, the cells were washed three times with ice-cold PBS and incubated with Alexa Fluor_488 goat anti-rabbit secondary antibody (1:50 dilution) for 2 h at room temperature. The stained cells were then washed three times with PBS, mounted on slides using fluoroshield mounting medium with DAPI  and observed with an AxioVision 4 imaging system . 5/well) were seeded into a 48-well plate and incubated for 24 h. Confluent cell cultures were scratched using a sterile pipette tip and then washed with PBS. After incubation for 8 h, the cells which migrated into the wound region were counted and captured using an Olympus CKX53 inverted microscope .For the wound healing assays, the cells . A p value of less than 0.01 was considered to be statistically significant.All the data were presented as the mean ± SD of at least three independent experiments. The significance of the difference between the two paired groups was analyzed using a Student’s In conclusion, our results show that we have successfully established three cisplatin-resistant human OSCC cell lines: YD-8/CIS, YD-9/CIS, and YD-38/CIS. These cell lines have highly similar biological characteristics, such as acquired drug resistance and EMT induction, and could be used as ideal models for further studies on the reversal of cisplatin-resistance in OSCC. They can also be used to help clarify the mechanism behind the molecular changes occurring in chemoresistance.


OSF-2 transcript , protein expression in cell lysates and cell culture medium (western blot), and expression of the OSF-2 protein in cell lines (immunofluorescence) were investigated in this study. Increased expression of OSF-2 mRNA was observed in drug-resistant cells and followed by increased protein expression in cell culture media of drug-resistant cell lines. A subpopulation of ALDH1A1-positive cells was noted for W1DR and W1MR cell lines; however, no direct co-expression with OSF-2 was demonstrated. Both drugs induced OSF-2 expression after a short period of exposure of the drug-sensitive cell line to DOX and MTX. The obtained results indicate that OSF-2 expression might be associated with the development of DOX and MTX resistance in the primary serous W1 ovarian cancer cell line.One of the main obstacles to the effective treatment of ovarian cancer patients continues to be the drug resistance of cancer cells. Osteoblast-Specific Factor 2  is a secreted extracellular matrix protein (ECM) expressed in fibroblasts during bone and teeth development. Expression of OSF-2 has been also related to the progression and drug resistance of different tumors. The present study investigated the role of OSF-2 by evaluating its expression in the primary serous ovarian cancer cell line, sensitive (W1) and resistant to doxorubicin (DOX) (W1DR) and methotrexate (MTX) (W1MR). The  Chemoresistance of cancer cells—inherent or developed during treatment—remains an obstacle to the effective treatment of ovarian cancer patients . Hundredwww.ensembl.org, ENSG00000133110) with a molecular mass from 83.5 to 93.5 kDa [Osteoblast-Specific Factor 2 (OSF-2), also known as a Periostin (POSTN), is a secretory glycoprotein expressed as at least six different isoforms , with statistically higher expression in chemoresistant, high-grade serosum and endometrioid tumors than in chemosensitive ones. In vitro experiments proved that OSF-2 stimulated the resistance of ovarian cancer cells to carboplatin and PAC . AnotherTo investigate the role of OSF-2 in drug resistance development, we used a model of ovarian cancer, the most lethal gynaecological malignancy . At the Previously, from the drug-sensitive W1 ovarian cancer cell line, we have developed a series of cell lines resistant to CIS, PAC, DOX, and topotecan (TOP)—drugs used in ovarian cancer chemotherapy, as well as vincristine (VIN) and MTX, which are not used in standard ovarian cancer treatment . These cOSF-2 gene in the following ovarian cancer cell lines was examined: high-grade serous—OVCAR3, PEA1, PEA2; low-grade serous—PEO23; serous—SKOV-3; endometroid adenocarcinoma - A2780 and primary ovarian cancer cell line—W1. The highest OSF-2 gene expression was observed in the A2780 cell line, and was assigned as 1. In comparison, for the W1 cell line, about a 20-fold lower expression level was noted. In the consecutive investigated cell lines, expression levels were much lower. Due to big differences in expression level among the examined cell lines, the results are presented as a logarithmic scale  increased levels of the OSF-2 transcript in both cell lines  in response to DOX treatment with a maximum increase in transcript level of about 3.5-fold after 72 h of treatment (OSF-2 transcript level after 72 h of treatment (p < 0.05) with a maximum increase in transcript level of about 1.7-fold  and MTX (50 and 100 ng/mL) for 24, 48, and 72 h. Then, changes in gene expression were investigated. The dose- and time-dependent increase in reatment A. A simi1.7-fold B was obsPreviously, we observed an ALDH1A1 CSCs-like population in PAC- and TOP-resistant ovarian cancer cell lines ,42. In tPreviously in drug-resistant cell lines, the increased expression of ECM molecules, like COL3A1, COL21A1, and LOX in ALDH1A1-positive cells was observed ,43. TherChemoresistance of cancer cells still remains one of the obstacles to the effective treatment of ovarian cancer patients The cellular mechanisms of resistance are already well-described in the literature. Cancer-tissue-specific mechanisms are still being investigated and need further evaluation. This is important especially from a clinical point of view, as it was hypothesized that the tumor microenvironment is a dominant force in multidrug resistance and low treatment efficacy of cancers . These mUsing the RNA microarray technique, we have analyzed the expression of ECM molecules in ovarian cancer cell lines resistant to CIS, PAC, DOX, TOP, VIN, and MTX. The obtained results found OSF-2 to be overexpressed in cell lines resistant to DOX and MTX . The eleOSF-2 in A2780 endometroid adenocarcinoma [OSF-2 expression [OSF-2 transcript levels in DOX- and MTX-resistant W1 cell lines. Since PCR primers used in our study recognize all of the OSF-2 isoforms, a more detailed western blot analysis was performed. As shown in The expression analysis conducted for the different ovarian cancer cell lines confirmed the very high expression of arcinoma , whereaspression , we founOSF-2 expression after a short period of treatment of a sensitive OC cell line with DOX or MTX. In a similar experiment conducted by Xiao et al., increased expression of periostin in SW480 and HT-29 colon cancer cell lines treated with oxaliplatin and fluorouracil was demonstrated for 24 to 72 h [As previously reported, most studies on the effects of cytotoxic drugs describe the difference in gene expression between drug-sensitive and resistant cell lines. The results of our previous experiments indicate that the expression of some genes changes after a short exposure time of cells to cytotoxic drugs ,52,53. I to 72 h . The resDifferent models of drug resistance development are described in the literature. In the last 10 years, one of the most-studied models of drug resistance is the one based on the theory of CSCs . SeveralOSF-2 expression seems to play a role in drug resistance—not only in vitro, but also in vivo. In two independent ovarian cancer studies, periostin expression in tumor stroma was associated with chemotherapy resistance. In a study by Sung et al., stromal OSF-2 expression was significantly higher in platinum-resistant patients, and correlated with the FIGO stage and tumor recurrence rate after the first treatment. Patients with high OSF-2 expression in stroma also had significantly lower overall survival (OS) and progression free survival (PFS) than those with low expression. In contrast, expression in tumor cells did not correlate with patients’ survival; however, patients with high OSF-2 expression in both compartments had shorter OS and PFS. . In a stTaken together, periostin seems to play a role in drug resistance, tumor growth, and metastasis in many cancers. As metastatic tumors are usually much more resistant to chemotherapy, we can suppose that periostin could also play a role in the drug resistance of metastatic tumors, such as in the case of drug-resistant cell lines. However, we did not find any data comparing resistant profiles of primary and metastatic tumors together with OSF-2 expression.®488 Donkey Anti-Rabbit and Alexa Fluor®594 Donkey Anti-mouse IgG—were obtained from Jackson ImmunoResearch Laboratories . Western blot reagents  were purchased from Biorad .Culture media , fetal bovine serum, antibiotic-antimycotic solution, L-glutamine, sodium private, DAPI mounting medium, and DOX and MTX were purchased from Sigma . Rabbit polyclonal anti-OSF-2 Ab was obtained from Proteintech , rabbit monoclonal anti-ALDH1A1 Ab was purchased from Abcam , mouse monoclonal anti-ALDH1A1 Ab was purchased from ABGENT , and rabbit polyclonal anti-GADPH Ab was purchased from Santa Cruz Biotechnology . Goat policlonal anti-rabbit horseradish peroxidase (HRP)-conjugated Ab was purchased from Cell Signaling . The fluorescent secondary antibodies—Alexa FluorThe human ovarian carcinoma A2780, SKOV-3, and OVCAR-3 were purchased from ATCC . The human ovarian carcinoma PEA1, PEA2, and PEO23 were purchased from Sigma . The human primary ovarian cancer cell line W1 was established from the tumor tissue of an untreated 54-year-old Caucasian female patient diagnosed for serous ovarian adenocarcinoma . Cells grow as a monolayer, and present an epithelial morphology and adherent growth model. The W1 subline, resistant to MTX (W1MR) and DOX (W1DR) were obtained by exposing W1 cells to the MTX or DOX at incrementally increasing concentrations. The final concentrations used for selecting the resistant cells were 28 ng/mL of MTX, and 100 ng/mL of DOX. The increase in resistance according to the parental drug-sensitive cell line W1 was as follows: 138-fold for W1MR vs. W1, and 10.3-fold for W1DR vs. W1 .2.Cells were cultured in Minimum Essential Medium Eagle (MEM) medium (A2780), RPMI-1640 medium (W1), RPMI-1640 medium supplemented with 2mM sodium private , or Dulbecco’s Modified Eagle Medium (DMEM) (SKOV-3) supplemented with 10% fetal bovine serum, 200 mML-glutamine, penicillin (100 units/mL), streptomycin (100 units/mL), and amphotericin B (25 μg/mL) at 37 °C in an environment of 5% COThe total RNA was extracted from cells using a Gene Matrix Universal RNA purification Kit  according to the manufacturer’s protocol. Reverse transcription was performed using the M-MLV reverse transcriptase  and a thermal cycler  as described in the manufacturer’s protocol. The cDNA was amplified from two µg of RNA. Real-time PCR was performed using the 7900 HT Fast Real-Time PCR System , Maxima SYBR Green/ROX qPCR Master Mix , and the sequence-specific primers, as indicated in Ct−ΔΔ (where ΔΔCt = ΔCt of the sample (drug-resistant line) − ΔCt of the calibrator (drug-sensitive line)). The graphs were plotted using Sigma Plot.The relative quantification (RQ) method was used for gene expression analysis, where RQ estimates the difference at the level of gene expression against the calibrator . As a calibrator, the drug-sensitive cell lines (A2780 or W1) were used. The standard formula was applied: RQ = 26 cells/25 μL) were prepared using a RIPA buffer containing a protease inhibitor cocktail , and then, supernatant was collected after centrifugation at 4 °C and 12,000× g for 15 min. A Bradford protein assay system  was used to determine the protein concentrations. The isolation of proteins from culture media was prepared after a 72 h culture of cells in serum-free media. Next, the media was centrifuged at 15,000 rpm for 30 min at RT, and supernatants were placed in Amicon Ultra-15 3K centrifuge filter devices  and centrifuged using a swinging-bucket rotor for 60 min at 4,000× g/RT.Cell lysates . Next, the membrane was incubated with rabbit policlonal anti-OSF-2 or rabbit monoclonal anti-ALDH1A1 antibodies at dilutions of 1:500, followed by incubation with the appropriate HRP-conjugated secondary antibody. The bands detection was analyzed using a chemical luminescence kit  and Hyperfilm ECL . The quality of protein loading was made by reblotting the membrane with a rabbit anti-GAPDH antibody (1:1000) and goat anti-rabbit HRP-conjugated antibody For the western blot analysis, 10 μg protein from each sample were resuspended in 4× loading buffer  and loaded into each well of a 4–20% mini-PROTEAN®488 secondary antibody  for 1 h at room temperature was proceed. In the double immunofluorescence, the cells were incubated with the mixture of two respective green dye-labelled  and red dye-labelled  secondary antibodies for 1 h at room temperature. Finally, cells were washed with PBS and mounted in DAPI mounting medium. The expression analysis and pictures were taken under fluorescence microscope .For immunofluorescence and double immunofluorescence analysis, cells were cultured in 24-well chamber glass slides and grown to a near-confluent state. The standard procedure was performed as follows. The culture medium was replaced by PBS for rinsing, and next by ice-cold acetone/methanol (1:1) for 10 min for fixation and permeabilization. Then, cells were washed with PBS and blocked in 3% BSA for 30 min at room temperature. After that, the incubation with primary antibodies was prepared. For classic immunofluorescence one type of primary antibody at a time was used . For double immunofluorescence purpose the cells were incubated with the mixture of two primary antibodies simultaneously: OSF-2  + ALDH1A1 . After 2 h incubation at room temperature, cells were washed with PBS and incubated with secondary antibodies. For single immunofluorescence, the incubation with green dye-labelled Alexa FluorOSF-2 was measured after a short period of exposure to a citotoxic agent. The drug-sensitive cell line (W1) was cultured in 1 mL of medium at a density of 0.5 × 106 cells per well (six-well plate). Then, the DOX at 15 and 20 ng/mL concentrations or MTX at 50 ng/mL and 100 ng/mL concentrations were added, respectively . The int-test. p < 0.05 was considered to indicate a statistically significant difference.Data are presented as standard error of the mean (SEM) and were analyzed using Student’s To our knowledge, this study is the first to describe the expression analysis of OSF-2 in drug-resistant ovarian cancer cell lines. Our research showed elevated levels of OSF-2 expression in W1 DOX- and MTX-resistant ovarian cancer cell lines and corresponding media. The short exposure time of cells to DOX- or MTX-induced expression of OSF-2 in the drug-sensitive cell line, which may suggest its potential role in the primary resistance of this cell line before other mechanisms of drug resistance appear. An extracellularly secreted OSF-2 protein could be involved in CAM-DR. Thus, we have found that the expression of OSF-2 might be associated with the development of DOX and MTX resistance of the primary serous W1 ovarian cancer cell line.


Endothelial cell senescence is a hallmark of vascular aging that predisposes to vascular disease. We aimed to explore the capacity of the renin–angiotensin system (RAS) heptapeptide angiotensin (Ang)‐(1‐7) to counteract human endothelial cell senescence and to identify intracellular pathways mediating its potential protective action. In human umbilical vein endothelial cell (HUVEC) cultures, Ang II promoted cell senescence, as revealed by the enhancement in senescence‐associated galactosidase  positive staining, total and telomeric DNA damage, adhesion molecule expression, and human mononuclear adhesion to HUVEC monolayers. By activating the G protein‐coupled receptor Mas, Ang‐(1‐7) inhibited the pro‐senescence action of Ang II, but also of a non‐RAS stressor such as the cytokine IL‐1β. Moreover, Ang‐(1‐7) enhanced endothelial klotho levels, while klotho silencing resulted in the loss of the anti‐senescence action of the heptapeptide. Indeed, both Ang‐(1‐7) and recombinant klotho activated the cytoprotective Nrf2/heme oxygenase‐1 (HO‐1) pathway. The HO‐1 inhibitor tin protoporphyrin IX prevented the anti‐senescence action evoked by Ang‐(1‐7) or recombinant klotho. Overall, the present study identifies Ang‐(1‐7) as an anti‐senescence peptide displaying its protective action beyond the RAS by consecutively activating klotho and Nrf2/HO‐1. Ang‐(1‐7) mimetic drugs may thus prove useful to prevent endothelial cell senescence and its related vascular complications. ACE2angiotensin‐converting enzyme 2Ang‐(1‐7)angiotensin‐(1‐7)AngangiotensinAREantioxidant‐regulated elementsBSAbovine serum albuminFCSfetal calf serumHO‐1heme oxygenase‐1ICAM‐1intercellular adhesion molecule‐1IL‐1βinterleukin‐1βNrf2nuclear factor‐erythroid 2‐related factor 2RASrenin–angiotensin systemr‐klothorecombinant klothoSA-β-galsenescence‐associated β‐galactosidaseSASPsenescence-associated secretory phenotypeSn‐PPtin protoporphyrin IXTIFstelomere dysfunction‐induced fociTRF‐1telomere protein telomeric repeat binding factorVCAM‐1vascular cell adhesion molecule‐1γH2AXphosphorylated histone H2AX1Vascular aging is a complex multifaceted process displaying functional and structural alterations that ultimately favor vascular disturbances, including endothelial dysfunction and atherosclerosis  plays a pivotal role in the regulation of cardiovascular homeostasis, in both health and disease. In recent years, a number of studies have been dedicated to decipher the biological actions of novel RAS components, such as angiotensin (Ang)‐(1‐7). This heptapeptide is generated not only from angiotensin II (Ang II) through the action of angiotensin‐converting enzyme 2 (ACE2), but also from angiotensin I (Ang I) via neutral endopeptidase activity  has been regarded as a physiological antagonist of Ang II by opposing its vasoconstrictor, proliferative, hypertrophic, or pro‐inflammatory actions /antioxidant‐regulated element (ARE) system is a major evolutionary conserved cytoprotective system. It is also nowadays considered a powerful modulator of species longevity /Mas receptor axis to antagonize cell senescence induced by Ang II and by other non‐RAS stressors. The participation of klotho and Nrf2/HO‐1 in the protective actions of Ang‐(1‐7) on endothelial cells was also analyzed.22.1The fraction of senescence‐associated β‐galactosidase  cells, used as a marker of cell senescence, was of 2.97 ± 0.23% in control unstimulated human umbilical vein endothelial cell (HUVEC) pre‐senescent cultures. After the exposure to Ang II 100 nM) for 18 hr, the fraction of cells positively stained for SA‐β‐gal was significantly enhanced, and this effect was mitigated by Ang‐(1‐7)  at the sites of injury  in late‐passage (P12) senescent HUVEC, in which the proliferation rate was markedly reduced and the average SA‐βgal+ staining in basal unstimulated conditions was shifted up to 53.52 ± 3.44%  has been proposed as a physiological antagonist of Ang II. However, we next aimed to determine the capacity of the heptapeptide to counteract endothelial senescence induced by other stressors independent of the RAS. The pro‐inflammatory cytokine interleukin (IL)‐1β was chosen based on its growing relevance in human vascular disease Libby, . Similar2.3Senescent endothelial cells undergo a series of changes that result into an activated pro‐inflammatory and secretory phenotype that eventually favors leukocyte recruitment.Consistent with this notion, both Ang II and IL‐1β enhanced the expression of the adhesion molecules, intercellular adhesion molecule (ICAM)‐1 Figure a,d and v2.4To analyze the cytoprotective mechanisms that might be on the basis of the anti‐senescence action of Ang‐(1‐7), we first explored a possible role for klotho. In endothelial cells, human recombinant klotho (r‐klotho) prevented the enhanced SA‐β‐gal+ cell number induced by both Ang II and IL‐1β, without affecting cell senescence by itself Figure a. In fac2.5To gain insight into the common cytoprotective pathways that might be activated by both Ang‐(1‐7) and klotho, we next focused on Nfr2/HO‐1. First, the pharmacological activation of Nrf2 by means of sulforaphane protected endothelial cultures against cell senescence induced by both Ang II and IL‐1β Figure a. MoreovHO‐1 levels were equally enhanced by either Ang‐(1‐7) or r‐klotho Figure e,f. In fGlobally considered, these data identified Nrf2/HO‐1 as key mediators of the cytoprotective effects derived from the consecutive activation of Ang‐(1‐7) and klotho. This concept was further reinforced using a functional ex vivo vascular reactivity assay. In noradrenaline pre‐contracted murine mesenteric microvessels, the vasodilation exerted by Ang‐(1‐7), but also the one induced by r‐klotho, was markedly inhibited by Sn‐PP, while the one exerted by the vasodilator acetylcholine remained unaffected Figure k.3Vascular aging is a main risk factor for developing adverse cardiovascular events /Mas axis, may be a suitable target to delay endothelial cell senescence, one of the key hallmarks of vascular aging that drives to endothelial dysfunction and atherosclerosis.We herein demonstrate that Ang‐(1‐7) attenuates total and telomeric DNA damage, an early senescence‐associated event that signals downstream to provoke growth arrest and senescence , klotho is able to exert nitric oxide‐dependent vasorelaxant actions  as a valuable tool to protect against human endothelial cell senescence through the consecutive activation of klotho and the Nrf2/HO‐1 axis. Over the last years, a notable effort is being made to develop pharmacological activators of the ACE2/Ang‐(1‐7)/Mas axis to fight a number of conditions, including cardiovascular diseases. In light of the present results, these drugs may prove useful to counteract endothelial cell senescence and premature vascular aging, as well as their deleterious complications. Since blocking senescence with Ang‐(1‐7) might promote dysplasia or neoplasia of endothelial cells, or Ang‐(1‐7) may itself exert hypotension or cardiac and renal fibrosis . M199 medium, fetal calf serum (FCS), and trypsin‐EDTA were from Biological Industries . Human r‐klotho and IL‐1β were purchased from Abcam  and Peprotech , respectively. Ang‐(1‐7) and the Mas antagonist A779 were purchased from Bachem . Sn‐PP and sulforaphane were from Frontier Scientific  and LKT Laboratories , respectively. All other reagents were purchased from Sigma  unless otherwise stated.4.22. For experiments, pre‐senescent cells at passages 1–5 were incubated for the indicated time periods with the different test compounds in M199 medium supplemented with 10% FCS, ECGS, heparin and antibiotics. For some experiments, senescent HUVEC were used at passage 12. All the procedures were reviewed and approved by the ethics committee of Universidad Autónoma of Madrid and Hospital Universitario La Paz, respectively, and written informed consent was obtained from all cord donors.Human umbilical vein endothelial cells (HUVEC) were isolated from umbilical cords, as previously described  as previously described  were examined by immunofluorescence microscopy as previously described  were then perfused across the endothelial monolayer. In all experiments, leukocyte interactions were determined after 5 min at 0.5 dyn/cm2. Cells interacting with the surface of the endothelium were visualized and recorded  using phase‐contrast microscopy .Mononuclear cells were obtained from buffy coats of healthy donors by Ficoll‐Hypaque density gradient centrifugation, as previously described  according to the manufacturer instructions.After the appropriate treatments and incubation times, supernatants were collected, centrifuged at 900 4.8Klotho, Nrf2, and HO‐1 levels in HUVEC were determined by Western blot as previously described  were mounted on a small‐vessel myograph to measure isometric tension as described before (Peiró et al., 4.12t test and one‐way ANOVA followed by Bonferroni post hoc test or two‐way ANOVA, as appropriate. A p value ≤0.05 was considered statistically significant.Results are expressed as mean ± SEM. Statistical analysis was performed using Student's None declared.C.P., C.F.S.‐F., and T.R. conceived the study. A.S.‐H.L., A.R., L.A.V., S.V., I.V., P.M. R.L., N.P.‐L., and I.S.‐P. performed in vitro and ex vivo experiments, analyzed raw data, performed statistical analysis, and drew figures. J.D.E. and M.J.S. designed and analyzed the DNA damage foci and flow chamber experiments, respectively. C.P., C.F.S.‐F., and T.R. wrote the manuscript. J.L.B., J.D.E., and M.J.S. contributed to the discussion. All authors reviewed and approved the final version of the manuscript. Click here for additional data file.