Osteosarcoma is the most common primary malignant tumour of bone occurring in children and young adolescents and is characterised by complex genetic and epigenetic changes. The miRNA miR-486-5p has been shown to be downregulated in osteosarcoma and in cancer in general.mir-486 locus is epigenetically regulated, we integrated DNA methylation and miR-486-5p expression data using cohorts of osteosarcoma cell lines and patient samples. A CpG island in the promoter of the ANK1 host gene of mir-486 was shown to be highly methylated in osteosarcoma cell lines as determined by methylation-specific PCR and direct bisulfite sequencing. High methylation levels were seen for osteosarcoma patient samples, xenografts and cell lines based on quantitative methylation-specific PCR. 5-Aza-2′-deoxycytidine treatment of osteosarcoma cell lines caused induction of miR-486-5p and ANK1, indicating common epigenetic regulation in osteosarcoma cell lines. When overexpressed, miR-486-5p affected cell morphology.To investigate if the miR-486-5p represents a highly cancer relevant, epigenetically regulated miRNA in osteosarcoma, and this knowledge contributes to the understanding of osteosarcoma biology.The online version contains supplementary material available at 10.1186/s12864-022-08346-6. High-grade osteosarcoma is the most prevalent primary malignant tumour of bone, affecting both children, adolescents and, more rarely, elderly people. Following the introduction of multi-agent chemotherapy in the 1970s, the 5-year survival rate increased considerable reaching 60–70% among patients with conventional high-grade osteosarcoma , and alsOsteosarcoma is characterized by considerable phenotypic and genomic heterogeneity, and few recurrent targetable genetic changes have been reported. Osteosarcoma exhibits a complex karyotype with high genetic and chromosomal instability seen as multiple rearrangements across the genome, kataegis and chromothripsis –6. The gEpigenetic mechanisms are fundamental drivers of tumour initiation, development and progression . SeveralMicroRNAs (miRNAs) are a key class of epigenetic regulators as they act to post-transcriptionally silence large numbers of genes without modifying the DNA . miRNAs mir-486 locus in detail. Finally, the in vitro effect of miR-486-5p overexpression on cell morphology and proliferation was investigated.In an effort to advance the understanding of osteosarcoma biology, we aimed to investigate if the expression of miR-486-5p was epigenetically regulated through methylation of promoter regions. We analysed miR-486-5p expression and DNA methylation levels in a cohort of osteosarcoma cancer cell lines and patient samples. Qualitative and quantitative methylation analyses were performed, allowing us to characterize the n = 9), osteosarcoma cell lines (n = 17) and normal bone samples (n = 6) by quantitative real-time reverse-transcription PCR (qRT-PCR). The mean expression of miR-486-5p was reduced 3-fold in patient samples (p < 0.01) and 300-fold in cell lines (p < 0.0001) compared to normal bone  gene. Several transcript variants of human ANK1 exist, with multiple transcriptional start sites (TSS), promoter regions and CGIs. The ANK1 variants 1–4 represents medium-sized transcripts, ANK1 variant 9 is a longer transcript while sANK1 represents the short variants 5, 7 and 10  and normal bone samples (n = 4) using Illumina HumanMethylation27 BeadChips. This microarray contains one CpG site in the CGI CpG79  at the TSS (position chr8:41655140) of ANK1 (variant 1–4). The average difference of methylation in cell lines versus normal bone samples was 0.36 (DeltaBeta) for CpG79, providing a first evidence for methylation of the mir-486 locus.p < 0.0001) and the methylation was higher in cell lines than in bone (p < 0.01). Taken together, this indicated an association between low expression of miR-486-5p and CpG methylation of ANK1  were treated with the demethylation agent 5-Aza-2′-deoxycytidine (5-Aza). After 72 h, the relative expression levels of miR-486-5p in untreated and treated cell lines were quantified by qRT-PCR. A significant difference between the cells before and after treatment was observed (p = 0.02). miR-486-5p showed at least a 1.3-fold (30%) induction in 8/12 tested cell lines  before 5-Aza treatment and r = 0.92 (p < 0.0001) after treatment of the cell lines  was quantified by qRT-PCR. pri-mirR486 showed at least 1.3 fold (30%) induction in 9/12 cell lines after 5-Aza treatment, confirming the epigenetic effects seen on the mature miRNA  could be observed across the cell line panel if one of the cell lines, MG-63, was omitted . The cell line MG-63 showed very high expression levels of ANK1 variants 1–4, however as the sample seems to be a biological and not a technical outlier, it was not removed from the dataset.In osteosarcoma cell lines, ANK1 variant 1–4 expression was induced in 8/12 cell lines upon 5-Aza treatment  could be seen between the fold change observed for ANK1 and the fold change observed for miR-486-5p upon Aza treatment across all 12 cell lines  analysis was done for CGI CpG79 and CpG171, both located upstream of nes Fig. . InteresDirect bisulfite sequencing was performed on osteosarcoma cell lines. The analysis was done on an extended region of the CGI CpG79, covering a total of 34 CpGs including the region for the MSP on CpG79 Fig. . The resmir-486 was analysed quantitatively by quantitative methylation-specific polymerase chain reaction (qMSP) in a larger cohort of samples, including osteosarcoma cell lines (n = 20), xenografts (n = 41), patient samples (n = 14) and bone (n = 5). Methylated percent of methylated reference (PMR) was set to > 5.2. High qMSP methylation pattern was only observed in cancer samples, comprising both cell lines, xenografts and patient samples . For the cell lines, 17/20 (85%) showed increased methylation (>2x) compared to the bone average. However, increased methylation was also observed for 8/14 (60%) of the patient’s samples and 17/41 (40%) of the xenografts. Together, these observations suggest that the CGI CpG79 is highly methylated in osteosarcoma.The methylation of a region of CpG79 of p = 0.2). However, a clear change in cell morphology was observed in all three cell lines where cells became flatter, rounder and smaller  repressed both ANK1 and miR-486-5p [It has been shown that alternative promoters give individualized regulation of transcripts with distinct first exons, providing an expression pattern with a strong tissue preference . The regid cells , and hasid cells , 34. Theiant 1–4 . An earl variant . The sANR-486-5p , and wasThe induction of miR-486-5p expression in osteosarcoma cells resulted in a change in cell morphology, as well as a pattern of reduced cell proliferation in 2 out of 3 cell lines tested. A morphological change in MG-63 cells following overexpression of miR-486-5p has been described previously, possibly related to an EMT-like phenotype . Throughmir-486 in various tissue types would be highly interesting.There were a couple limitations to our study. The sample size of the normal samples is smaller than the included osteosarcoma samples. Furthermore, the manuscripts only showed that the hypermethylation in the differentially methylated region correlated with downregulation of miR-486-5p expression. Although a direct effect is not proven, an inverse pattern between methylation and gene expression is shown and the other results performed support the observation that the miRNA is epigenetic regulated. Further investigations regarding the epigenetic regulation of ANK1 variant 1–4. This implies a tumor suppressive role of miR-486-5p in osteosarcoma, and the findings may lead to clarify the tissue/disease-specific regulation of the miR-486-5p expression. The main effect of miR-486-5p overexpression was morphological changes in osteosarcoma cells. The epigenetic regulation should be further correlated with clinical characteristics, and the functional role needs to be further validated in extended model systems.In conclusion, the present study of miR-486-5p shows that the low expression observed in osteosarcoma is caused by cancer-specific methylation of the upstream promoter region of n = 21) composed of 143B, CAL-72, G-292, HAL, HOS, IOR/MOS, IOR/OS9, IOR/OS10, IOR/OS14, IOR/OS15, IOR/OS18, IOR/SARG, KPD, MG-63, MHM, MNNG/HOS, OHS, OSA, Saos-2, U-2 OS and ZK-58 were obtained from ATCC (www.lgcstandards-atcc.org), DSMZ (http://www.dsmz.de/) or research partners in the EU funded EuroBoNeT project [A panel of human osteosarcoma cell lines ( project . These c project  and authn = 19) and at the University of Valencia (n = 22) [The xenograft cohort comprised osteosarcoma xenografts established at the Norwegian Radium Hospital ((n = 22) , 53. In (n = 22) .The osteosarcoma patient sample panel comprised fresh-frozen tissue from 14 high-grade osteosarcoma samples collected at the Norwegian Radium Hospital. The tumours were diagnosed by an osteosarcoma pathologist, according to the current World Health Organization classification .n = 2) or obtained from amputations of cancer patients at the Norwegian Radium Hospital (n = 4), where bone samples were collected distant from the tumour margin. DNA copy number analysis of the latter four samples (Bone 1–4) showed normal diploid karyotype [Six bone samples were used as normal controls. Normal bones were purchased from Capital Biosciences   compliant data can be downloaded from the GEO repository (www.ncbi.nlm.nih.gov/geo/), accession number GSE28425.Total RNA from osteosarcoma cell lines and bones was extracted and quality controlled as previously described . miRNA eDNA was isolated using the Wizard Genomic DNA Purification Kit . For the initial analyses, DNA methylation profiling of 19 cell lines and four bone samples was performed using the Illumina HumanMethylation27 BeadChip , covering 27,000 CpG sites across the genome . For an The association between CpG methylation and miRNA expression data in osteosarcomas and normal samples was calculated using Pearson’s Correlation between miR-486-5p expression and methylation level (Beta) using the Methylation module v1.9.0 of GenomeStudio. DeltaBeta values for the groups of samples were calculated .ANK1 expression quantification, the Fast Cells-to-Ct reagents (Life Technologies) were used to generate cDNA. Transcript-specific TaqMan Gene Expression Assays (Life Technologies) were used for quantitative PCR. Gene expression was normalized towards RNU44 for miRNA quantification and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) for pri-miRNA hsa-mir-486-1 and ALK1 quantification. Overview of TaqMan assays are found in Additional file T method.qRT-PCR was performed using the ABI PRISM 7500 DNA Sequence Detection System . TaqMan MicroRNA Reverse Transcription Kit and TaqMan MiRNA Assays (Life Technologies) were used to generate cDNA and to quantitatively detect the expression of mature miR-486-5p. For pri-miRNA hsa-mir-486-1 and Osteosarcoma cells were cultured in the presence of 1 μM 5-Aza , as previously described . The celGenomic DNA from tumour and normal samples was isolated by standard phenol chloroform extraction or using the Wizard Genomic DNA Purification Kit (Promega). DNA (1.3 μg) was bisulfite treated using the EpiTect Bisulfite Kit (Qiagen) and purified using the QIAcube (Qiagen).Primers for MSP were designed using Methyl Primer Express 1.0 (Life Technologies)  and flanked the MSP amplicons  were transiently transfected into osteosarcoma cells at a final concentration of 15 nM using the INTERFERin siRNA transfection reagent . The cellular growth after transfection was measured every 3 h, by a live-cell imaging system, IncuCyte ZOOM  and the percent of cells confluence was calculated with the corresponding software application (version 2013BRev1).p-values were derived from two-sided tests using a significance level of 0.05. Similarities between samples were calculated using Pearson’s correlation (r). Statistical differences between the group of cell lines before and after 5-Aza treatment were calculated using a paired t-test.Analyses were performed with the GraphPad Prism 9 software. Non-parametric Mann-Whitney U test was applied to compare groups of samples. Additional file 1: Supplementary Table S1. Overview of TaqMan assays for miRNA and mRNA qRT-PCR experiments. Supplementary Table S2. Primers used for qualitative Methylation-Specific Polymerase Chain Reaction (MSP), bisulfite sequencing (BS) and quantitative Methylation-Specific Polymerase Chain Reaction (qMSP). Supplementary Figure S1. Genomic overview of the mir-486/ANK1 locus. Supplementary Figure S2. Expression of ANK1 transcript variants in untreated and 5-Aza treated osteosarcoma cell lines. Supplementary Figure S3. Methylation level of miR-486 as assessed by methylation specific PCR (MSP).


The epidermal growth factor receptor (EGFR) is a therapeutic target in head and neck squamous cell carcinoma (HNSCC). Resistance to EGFR-targeted therapies, such as cetuximab, poses a challenging problem. This study aims to characterize acquired cetuximab resistance mechanisms in HNSCC cell lines by protein phosphorylation profiling. Through this, promising combination treatments can be identified to possibly overcome acquired cetuximab resistance in HNSCC. Protein phosphorylation profiling showed increased phosphorylation of Akt1/2/3 after cetuximab treatment in acquired cetuximab resistant cells compared to cetuximab sensitive cells, which was confirmed by western blotting. Based on this protein phosphorylation profile, a novel combination treatment with cetuximab and the Akt1/2/3 inhibitor MK2206 was designed. Synergy between cetuximab and MK2206 was observed in two cetuximab sensitive HNSCC cell lines and one acquired cetuximab resistant variant in simultaneous treatment schedules. In conclusion, this study demonstrates that increased Akt1/2/3 phosphorylation seems to be characteristic for acquired cetuximab resistance in HNSCC cell lines. Our results also show an additive to synergistic interaction between cetuximab and MK2206 in simultaneous treatment schedules. These data support the hypothesis that the combination of cetuximab with PI3K/Akt pathway inhibition might be a promising novel therapeutic strategy to overcome acquired cetuximab resistance in HNSCC patients. Head and neck squamous cell carcinoma (HNSCC) is the sixth most common cancer type worldwide and remains one of the most challenging malignancies to treat , 2. ThroOver the past decades, research has revealed that the epidermal growth factor receptor  plays an integral role in the tumorigenesis of HNSCC. Increased or sustained activation of the EGFR signaling pathway can induce malignant transformation through sustained signaling for cell proliferation, anti-apoptotic signaling, angiogenesis and metastasis . FurtherIn 2006, the anti-EGFR monoclonal antibody (mAb) cetuximab, was approved by the Food and Drug Administration (FDA) in combination with radiotherapy for locoregionally advanced HNSCC  of 4.1 years versus 2.4 years with radiotherapy alone) and in combination with platinum-based therapy and infusional 5-fluorouracil (EXTREME regimen) for the first-line treatment of patients with recurrent/metastatic (R/M) HNSCC  . In partThe reason for resistance to pembrolizumab treatment in HNSCC remains largely unknown. In this regard, several biomarkers, such as PD-L1 expression, immune infiltration, tumor mutational burden and immune-gene expression profiling, have been explored for their predictive potential. However, none of them could be validated in HNSCC so far . ConcernSens) HNSCC cell lines and their acquired cetuximab resistant (AcqRes) variants. Based on this protein phosphorylation profile, a novel combination treatment was designed to overcome acquired therapy resistance.Therefore, the present study aims to investigate a novel combination strategy to overcome acquired cetuximab resistance. We hypothesized that acquired cetuximab resistance in HNSCC may arise from the activation of compensatory signaling pathways following cetuximab treatment, which are able to reverse the inhibitory effects of cetuximab through phosphorylation of key proteins, thereby promoting cell survival. Pharmacological inhibition of these phosphorylated proteins might be essential to overcome acquired cetuximab resistance in HNSCC. Therefore, we first characterized the protein phosphorylation profile of two cetuximab sensitive (CetSens following cetuximab treatment for 168h using the colorimetric sulforhodamine B (SRB) assay. Isogenic AcqRes HNSCC cell lines (SC263-R and SCC22b-R) were previously generated by chronically exposing these initially CetSens cell lines  to cetuximab, starting with the IC50 concentration of cetuximab . The identity of each cell line was validated through short tandem repeat profiling .HPV-negative human HNSCC cell lines SC263 and SCC22b were kindly provided by Prof. Dr. Sandra Nuyts  and Prof. Dr. Olivier De Wever . These HNSCC cell lines were previously identified as Cetetuximab –14. In petuximab –14. All Sens and AcqRes cell lines after cetuximab treatment. The human phospho-kinase antibody array was performed according to the manufacturer’s protocol. In short, cells were lysed on 6-well plates after treatment with cetuximab . Twenty minutes before lysis, epidermal growth factor  was added to the medium. Protein concentrations were determined using the Pierce BCA protein kit . Next, samples were prepared and 450 μg of proteins were incubated overnight with nitrocellulose membranes which are spotted with 46 capture antibodies in duplicate. The specific target proteins  was used to determine the relative levels of protein phosphorylation in the Cetproteins . Only prIn order to validate findings of the human phospho-kinase antibody array with western blot, cell lysates were three times independently prepared as described above. Twenty micrograms of proteins were separated by SDS-page  and western blot was performed . Blocking of non-specific binding sites was done by incubation of the membrane with Odyssey blocking buffer TBS  for 1h at room temperature while shaking. Next, primary and secondary antibody incubation and washing was performed using the SNAP id 2.0 protein detection system (Merck Millipore) according to the manufacturer’s instructions. Membranes were incubated with the following antibodies: phospho-Akt (S473) rabbit mAb  and total Akt (pan) rabbit mAb . Anti-β-actin was used as an internal standard . Goat-anti-rabbit  or goat-anti-mouse  fluorescently labeled secondary antibodies were used and fluorescent detection was performed using the Odyssey imaging system (Li-Cor). Protein levels were quantified using Image Studio™ Lite, a software program available on the Odyssey imaging system. Phospho-Akt and Akt expression levels were corrected for loading differences based on beta-actin expression. Fold changes were calculated by dividing the mean fluorescent signal of the treatment and control groups.The baseline mRNA expression of Akt1/2/3 in HNSCC patients was examined using the RNA sequencing data from The Cancer Genome Atlas (TCGA) dataset . RNASeqV2 from TCGA was processed and normalized using the software package RNA-Seq by Expectation Maximization (RSEM) in order to generate transcripts per million. This dataset was downloaded from cBioportal.Cell survival was assessed using the colorimetric SRB assay, as previously described , 17. ThiCetuximab plus MK2206 with total treatment duration of 72hCetuximab for 168h with MK2206 added during the last 72h of treatment50 values (i.e. drug concentration causing 50% growth inhibition) were calculated using Graphpad Prism 9 software. Possible synergism between cetuximab and MK2206 was determined by calculation of the combination index (CI) using the Additive Model as described by others  and further dilutions were made in cell culture medium. ICy others –20. CI <We performed all experiments at least three times independently, unless otherwise stated. In cytotoxicity experiments, each condition was tested in triplicate in each of the three experiments. Differences in total and phosphorylated Akt1/2/3, determined with western blot, were statistically analyzed using the non-parametric Kruskal-Wallis test. Significant values were adjusted by the Bonferroni correction for multiple testing. One-way ANOVA with Tukey-Kramer HSD posthoc analysis was used to assess significant differences in Akt1/2/3 mRNA expression of HNSCC patients (RNASeqV2 TCGA data). The influence of cetuximab treatment on the effect of MK2206 alone was evaluated for each cell line using linear mixed models in case of non-independent observations. More specifically, combination treatment was set as fixed effect. A random intercept for experimental replicates was added in order to account for the dependence of observations between experiments. The influence of cetuximab resistance status on the effect of MK2206 alone was also evaluated using linear mixed models. Resistance status was set as fixed effect and a random intercept for cell line and experimental replicates was added to account for the dependence of observations between cell lines and experiments. GraphPad Prism 9 was used for data comparison and artwork. All statistical analyses were performed in JMP Pro 15 and SPSS v25. P-values below 0.050 were considered significant.Sens HNSCC cell lines and their isogenic AcqRes variants. Data analysis of the protein phosphorylation profile revealed a differential response of CetSens HNSCC cell lines and AcqRes variants to cetuximab treatment. As this change was most pronounced in substrates involved in the Akt signaling pathway, we focused further on the interpretation of these results in the next paragraph.In order to characterize acquired cetuximab resistance, a protein phosphorylation profile was established using the human phospho-kinase antibody array. Hereby, the effect of cetuximab treatment on the phosphorylation of various proteins (Sens HNSCC cell lines and AcqRes variants (Sens HNSCC cell lines (i.e. SC263-S and SCC22b-S) demonstrated decreased phosphorylation of Akt1/2/3 at T308 after cetuximab treatment. T308 is phosphorylated by phosphoinositide-dependent kinase-1 (PDK1) in the phosphoinositide 3-kinase (PI3K)/Akt pathway, leading to Akt activation  , proline-rich Akt substrate 40kDa (PRAS40) and WNK lysine deficient protein kinase-1 (WNK1)] in AcqRes variants, especially in SCC22b-R. This means that the Akt pathway is still activated in AcqRes cells following cetuximab treatment, leading to anti-apoptotic and pro-proliferative effects.The effects of cetuximab treatment on the phosphorylation of EGFR, Akt and other substrates involved in the Akt pathway were quantified in Cettivation . As a re(mTORC2) , was alsSens HNSCC cell lines and AcqRes variants . There was no statistical difference in the change of expression of total Akt1/2/3 following cetuximab treatment across cell lines , but not between SC263-S and SC263-R . This data demonstrated that there is a significant difference in mRNA expression between Akt1, Akt2 and Akt3 . As mentioned above, we previously identified CetSens cell lines based on the outcomes of experiments with a total treatment time of 168h  in CetSens HNSCC cell lines and the AcqRes variants  due to prolonged treatment duration. These fixed concentrations were based on the outcome of previous monotherapy experiments with a total treatment duration of 168h  was added during the last 72h of treatment. The goal of this prolonged treatment schedule was to decrease the used drug concentrations. In contrast to the first treatment schedule, Cet of 168h .Sens HNSCC cell lines and their AcqRes variants. The addition of MK2206 during the last 72h of treatment still resulted in a concentration-dependent cytotoxic effect of this compound. The IC50 values for MK2206 ranged from 1.073 ± 0.038 µM to 4.372 ± 1.182 µM . Furthermore, in this treatment regimen, the CI ranged from 0.578 to 0.867 , Akt1/2/3 (T308 and S473) and downstream substrates after cetuximab treatment. Although EGFR phosphorylation was decreased, Akt1/2/3 was still phosphorylated and activated following cetuximab treatment in AcqRes HNSCC cell lines. This was supported by the observed increase in phosphorylation of several downstream substrates of Akt, such as mTOR, p70S6 kinase, GSK-3α/β, PRAS40 and WNK1. Thus, in AcqRes HNSCC cell lines, the Akt pathway was still able to exerts its anti-apoptotic and pro-proliferative effects under cetuximab treatment, possibly leading to therapy resistance. Importantly, it is worth mentioning that the number of cell lines used in this study is a limiting factor. Therefore, our data needs to be validated in additional isogenic HNSCC cell lines  in order to strengthen the results. Although further confirmation in HNSCC cell lines and patient samples is still needed, based on our results, Akt represents a potential target to improve outcome of cetuximab-based treatment in HNSCC patients.Protein phosphorylation profiling showed a differential response of Cet2) to generate phosphatidylinositol 3,4,5-triphosphate (PIP3). The binding of PIP3 to Akt locates Akt to the plasma membrane and allows its phosphorylation and activation by PDK1. Akt can also be phosphorylated by other substrates and in response to cellular stress, such as ischemia, hypoxia and oxidative stress. The tumor suppressor phosphatase and tensin homolog (PTEN) catalyzes the dephosphorylation of PIP3 and is the major negative regulator of Akt signaling. Activated Akt exerts its effects by phosphorylating various downstream substrates, all resulting in anti-apoptotic or pro-proliferative effects  is characteristic for acquired cetuximab resistance in HNSCC cell lines. Importantly, this observation needs to be validated in a cohort of HNSCC patient samples in order to elucidate its clinical value. Nevertheless, previous research has already suggested that enhanced Akt activation can play a role in resistance to cetuximab, not only in HNSCC but also in colorectal cancer and non-small cell lung cancer –39. Incr3CA gene . For instivation . Accordification , 42. In  pathway , 44. Accectively , 42. Basand mTOR , 46. As Res HNSCC cell lines and Akt1, Akt2 and Akt3 are expressed in HNSCC patients, we hypothesized that the combination of cetuximab with an Akt inhibitor might be a potential novel therapeutic strategy to overcome acquired cetuximab resistance. To date, Akt inhibitors are not yet included in clinical practice , the Flemish cancer society (OZ7410). IDP and HZ are supported by research grants of Kom op tegen Kanker (Stand up to Cancer), the Flemish cancer society (OZ7410). We would like to thank Mr. Willy Floren for funding some of the equipment used in this study.JBV has had in the last three years consulting/advisory relationships with Immunomedics, Innate Pharma, Merck-Serono, Merck Sharp & Dome Corp, PCI Biotech, Synthon Biopharmaceuticals, Debiopharm, Cue Biopharma, and WntResearch and has received honoraria from Merck-Serono, MSD, and BMS.The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Mixed Lineage Leukemia (MLL/KMT2A) gene have a poor prognosis, especially when the disease strikes in infants. The poor clinical outcome linked to this aggressive disease and the detrimental treatment side-effects, particularly in children, warrant the urgent development of more effective and cancer-selective therapeutics. The aim of this study was to identify novel candidate compounds that selectively target KMT2A-rearranged (KMT2A-r) leukemia cells. A library containing 3707 approved drugs and pharmacologically active compounds was screened for differential activity against KMT2A-r leukemia cell lines versus KMT2A-wild type (KMT2A-wt) leukemia cell lines, solid tumor cells and non-malignant cells by cell-based viability assays. The screen yielded SID7969543, an inhibitor of transcription factor Nuclear Receptor Subfamily 5 Group A Member 1 (NR5A1), that limited the viability of 7 out of 11 KMT2A-r leukemia cell lines including 5 out of 7 lines derived from infants, without affecting KMT2A-wt leukemia cells, solid cancer lines, non-malignant cell lines, or peripheral blood mononuclear cells from healthy controls. The compound also significantly inhibited growth of leukemia cell lines with a CALM-AF10 translocation, which defines a highly aggressive leukemia subtype that shares common underlying leukemogenic mechanisms with KMT2A-r leukemia. SID7969543 decreased KMT2A-r leukemia cell viability by inducing caspase-dependent apoptosis within hours of treatment and demonstrated synergy with established chemotherapeutics used in the treatment of high-risk leukemia. Thus, SID7969543 represents a novel candidate agent with selective activity against CALM-AF10 translocated and KMT2A-r leukemias that warrants further investigation.Patients whose leukemias harbor a rearrangement of the  The conventional drug discovery path involves several drug development stages, from compound discovery, through to extensive preclinical drug optimization and characterization, which includes Absorption, Distribution, Metabolism, Elimination and Toxicity (ADMET) and efficacy studies in animal models, culminating in rigorous testing in humans through clinical trials. Due to this time-consuming process, it has typically taken more than a decade for new agents to advance from bench to bedside, with the vast majority of compounds failing to reach the final stages of clinical trial testing. As a result, an alternative and potentially more effective discovery approach based on drug repurposing has recently gained traction. Drug repurposing entails the use of drugs for indications other than those for which they were originally intended and therefore bypasses many development and optimization steps, shortening the timeline and decreasing the overall costs for drug approval.MLL gene, recently renamed KMT2A, present in approximately ten percent of patients with acute leukemia -approved drugs and pharmacologically active compounds with known targets against a KMT2A-r leukemia cell line with counter screening against a KMT2A-wildtype (KMT2A-wt) leukemia cell line. We identified SID7969543, an inhibitor of transcription factor Nuclear Receptor Subfamily 5 Group A Member 1 (NR5A1), as a novel selective candidate inhibitor against a subset of KMT2A-r and NR5A1 siRNA (M-003429-00-0010) targeting NR5A1 and On-TARGETplus control pool non-targeting scrambled siRNA (D-001810-10-20) were purchased from Millennium Science Pty Ltd.SID7969543 was purchased from Tocris Bioscience . 2-Cl-ATP was procured from Santa-Cruz Biotechnology . 2-CADO and cladribine were purchased from Sigma Aldrich . For the generation of lipid-based nanoparticles used to transfect leukemia cells with siRNA, cholesterol and the lipids 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG), and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-[lissamine rhodamine B sulfonyl] (DOPE-Rho) were obtained through Merck & Co., Inc. D-Lin-MC3-DMA was purchased from Assay Matrix Pty Ltd. siGENOME Human All cell lines used in this study were mycoplasma-free and have been authenticated using STR profiling. Cells were cultured as previously described , 8. Char®1280 library , Tocriscreen Plus library  and Selleck Inhibitor Library , together combining 3707 compounds. PER-485 (KMT2A-r) and CCRF-CEM  leukemia cells were seeded into 384-well culture plates. Test compounds were added to the assay plates for a final concentration of 5 μM (in DMSO stock). Plates were incubated for 72 hours at 37°C, 5% CO2. Resazurin was added and plates were incubated for 7 hours. The difference in relative fluorescence units at time zero and 7 hours of resazurin incubation was calculated for each well. The percentage cell viability for each test compound was calculated relative to the cells treated with vehicle (100% viability).The following chemical libraries were used in the screening as previously described : Prestwi50) was calculated as previously described . Quantitative PCR was run and analyzed on Applied Biosystem’s QuantStudio5 Real-Time PCR Detection System, using Taqman gene expression assays for genes NR5A1 (Hs00610436_m1), GUSB (Hs99999908_m1) and HPRT (Hs99999909_m1) Relative expressions were determined as previously described .Compound half-life was estimated by mouse liver microsomal stability assays as described .® instrument (Precision Nanosystems) as previously described (Lipid-based nanoparticles encapsulating siRNA targeting NR5A1 and scrambled siRNA (LNP-siRNA) were prepared using a NanoAssemblr™ Sparkescribed . Prior t6 cells/mL in 12 wells culture plates and incubated at 37°C, 5% CO2, with LNPs encapsulating anti-NR5A1 siRNA or scrambled siRNA at 250 nM siRNA concentration for up to 72 hours.Cells were seeded at 0.5 x 10KMT2A translocation found in infants and pediatric patients [t], and a KMT2A-wt leukemia cell line CCRF-CEM (in vitro models for high-risk disease. The viability of cells treated with a fixed concentration (5 µM) of the library compounds was assessed in resazurin-based cytotoxicity assays after a 72-hour incubation to allow selection of drugs acting rapidly on cell viability as previously described  in PER-485 cells, confirming our results from the original screen, it was less potent against the other KMT2A-r leukemia cell lines tested  that included the PER-485 and CEM cells used in the original screen, as well as four additional KMT2A-r cell lines (both ALL and acute myeloid leukemia (AML)) with either t (n=2) or t (n=2) CALM-AF10 translocated leukemias, which are KMT2A-wt but represent an aggressive leukemia subtype that shares underlying molecular etiological pathways with KMT2A-r leukemias, such as their dependency on DOT1L histone-lysine methyltransferase and an upregulation of HOXA cluster genes  KMT2A-r leukemia cells with IC50 values ranging from 2.5 to 5.9 µM . Corroborating our observations, previous studies have reported sensitivity of KMT2A-r ALL cells to nucleoside analogs , 18. To 50 < 10 µM) were leukemias with either a KMT2A or CALM-AF10 translocation , four out of six t and one t translocated KMT2A-r leukemia cell were sensitive to SID7969543, whereas the remaining two t KMT2A-r leukemia cells did not display sensitivity  and pyruvate kinase (PKM)  remained unchanged, demonstrating that SID7969543 hit its reported target   in sensise cells . Taken tWe next determined whether SID7969543 synergized with conventional chemotherapeutics used in the treatment of high-risk leukemia, namely, cytarabine, daunorubicin, etoposide, mitoxantrone, and topotecan Figure 4via the induction of apoptosis. In addition, the compound potentiates several standard-of-care chemotherapies used for treatment of high-risk leukemia. However, microsomal stability assays showed that SID7969543 has a short half-life and thus limited in vivo stability , a future larger scale screening in an expanded panel of KMT2A-r leukemia cell lines is warranted.SID7969543 preferentially inhibited the growth of a subset of KMT2A-r leukemia cells, which included various subtypes with different  subtype . While Kpathways –27. Thispathways –16 and fSID7969543 is an isoquinolinone derivative first identified in an ultra-high-throughput screen of approximately 65,000 compounds from the National Institute of Health’s Molecular Libraries Small Molecule Repository in a cell-based transactivation assay to search for potent and selective inhibitors of NR5A1 . NR5A1 iNR5A1 as a crucial gene for leukemic cell survival in one out of six primary AML samples  analysis with SID7969543 derivatives will be needed to identify the targeted pathway in KMT2A-r leukemia cells and develop clinically viable inhibitors. SAR analysis will pinpoint the functional groups essential for the KMT2A-r cell killing effect of the drug, guiding the development of analogs suitable for tagging and affinity chromatography to allow the identification of the molecular targets of SID7969543 and its derivatives in KMT2A-r leukemia cells. Silencing of candidate targets followed by downstream phenotypic analysis will subsequently provide insight into the identity of the true molecular target or targeted pathway for this class of compounds that mediates the observed KMT2A-r cell killing selectivity. These analyses will also direct the development of pharmacologically viable SID7969543 derivatives that have better drug-like properties and are suitable for evaluation in animal models of KMT2A-r leukemia.in vitro findings of our study demonstrating the rapid killing of a subset of KMT2A-r leukemia cells, particularly those arising in infants, support further study of SID7969543 to identify its molecular target, develop drug-like derivatives and use as a tool compound in vitro.While SID7969543 cannot be moved directly into the clinic, the strong KMT2A and CALM-AF10 translocated leukemia, warranting further investigation.Overall, our results show that SID7969543 represents a novel candidate agent with selective activity against The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.MKar conducted the majority of the experiments with technical support from AB and ER. MKar, LX, and KS analyzed the data. AM, TF, and GA executed the drug screening at the Drug Discovery Centre, Children’s Cancer Institute Australia. EM generated lipid nanoparticles for silencing experiments. LC and RK provided guidance and access to cell lines used in the study. MKav, MH, and MN provided guidance and support with study design. MKar, MJH, LX, and KS designed the experiments. MJH and KS conceived the project. MJH and KS supervised the project. KS, MKar, and LX wrote the manuscript. All authors contributed to the article and approved the submitted version.This research was supported by a grant from Cancer Council NSW (Program Grant PG-1601) and philanthropic funds from the Tenix Foundation. For this work, MKar was supported by a PhD topup scholarship by CRC Cancer Therapeutics. KS received funding from the Kids Cancer Alliance. LX was supported by early-career researcher fellowships from CINSW and Tour de Cure.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Few cell lines have been investigated in vitro, and previous results are inconsistent. Assuming that oxygen depletion accounts for the FLASH sparing effect, no sparing should appear for cells irradiated with low doses in normoxia.Irradiation with ultra-high dose rate (FLASH) has been shown to spare normal tissue without hampering tumor control in several Seven cancer cell lines  and normal lung fibroblasts (MRC-5) were irradiated with doses ranging from 0 to 12 Gy using FLASH (≥800 Gy/s) or conventional dose rates , with a 10 MeV electron beam from a clinical linear accelerator. Surviving fraction (SF) was determined with clonogenic assays. Three cell lines were further studied for radiation-induced DNA-damage foci using a 53BP1-marker and for cell cycle synchronization after irradiation.subclone. Similar cell cycle distributions and 53BP1-foci numbers were found comparing FLASH to CONV.A tendency of increased survival following FLASH compared with CONV was suggested for all cell lines, with significant differences for 4/7 cell lines. The magnitude of the FLASH-sparing expressed as a dose-modifying factor at SF=0.1 was around 1.1 for 6/7 cell lines and around 1.3 for the HeLain vitro. The magnitude of the FLASH effect differed between the cell lines, suggesting inherited biological susceptibilities for FLASH irradiation.We have found a FLASH effect appearing at low doses under normoxic conditions for several cell lines  Experimental evidence for the FLASH effect has been demonstrated 00 Gy/s, settings , as wellsettings .in vivo data available suggest that more sparing may occur in physoxic normal tissues than in severely hypoxic or nearly anoxic tumors  were acquired from American Type Culture Collection (ATCC). The human colon cancer cell line WiDr was acquired from LGC Promochem . The squamous cell carcinoma LU-HNSCC4 was established in our laboratory from a patient with a squamous cell carcinoma in the floor of the mouth . Cells wsubclone). Irradiation was performed under normoxic conditions at room temperature with the flasks lying flat and irradiated from beneath (beam angle 180 degrees). After irradiation, the flasks were returned to the incubator for 9-14 days. All flasks, including the non-irradiated controls, were terminated at the same occasion. Cells were fixed and stained with methylene-blue in 70% ethanol. Flasks were scanned using a flatbed scanner in 1,200 dpi resolution. Colony counts were performed with a standardized ImageJ-code  and manually checked. Surviving fraction (SF) was determined as the number of colonies with at least 50 cells divided by the number of plated cells (corrected for plating efficiency).Exponentially growing cells were trypsinized and plated in appropriate cell densities in 2.50 ml medium per Falcon T12.5 flask  and allowed to adhere overnight before irradiation. Control flasks for determination of the plating efficiency and the FLASH- and CONV-flasks were prepared identically on the same occasion, for each repetition. FLASH and CONV-flasks were irradiated minutes apart with doses from 0-9 Gy  and allowed to adhere overnight before irradiation with 3 Gy. At specific time points after irradiation, cells were washed with PBS and fixed with 4% paraformaldehyde for 20 min. After washing, cells were permeabilized with 0.5% Triton-X100 in PBS for 20 min, washed, blocked in blocking buffer  for 1 hour followed by 1 hour incubation with 53BP1 primary antibody (Invitrogen PA146147) and 1 hour of incubation with a secondary antibody (AlexaFluor anti-rabbit 488). Cell nuclei were counterstained with DAPI. Permeabilization, washing, blocking and staining steps were all performed at room temperature. Foci formation were assessed with a widefield fluorescence microscope, AxioOberver Z.1 , equipped with ×63/1.40 Plan-Apochromat oil-immersion objective lens and Colibri 7 solid state LED light source (Zeiss), and an ORCA-Flash4.0 V3 Digital CMOS camera . In each sample, at four different positions, fifteen Z-stack images were acquired, deconvoluted with a GPU-based deconvolution module and averaged using a Maximum Intensity Projection-algorithm . ImageJ was used for automated foci identification and quantification of DNA-double strand break (DSB) foci.subclone cells). At 24 h (6 h and 24 h for HeLasubclone cells), after irradiation cells were washed with PBS, harvested and fixated with ice-cold ethanol (70%). Cell nuclei were stained with propidium iodide  for 30 min at room temperature and DNA content was determined with an Accuri C6 Flow Cytometer . DNA-histograms were analyzed in ModFit LT 5.0 for Mac (BD Biosciences).150 000 - 500 000 cells were plated in 35 mm or 60 mm Petri Dishes  and allowed to adhere overnight before irradiation with 6 Gy (and 3 Gy for the HeLaIrradiation and dosimetry were performed as described previously . In summhttp://www.rstudio.com/) was used for statistical calculations. The parameters of the linear–quadratic model (2)) were fitted to the log (SF) using the nonlinear least-squares method (‘nls’ in RStudio). Two alternative models were fitted, one with separate α and β parameters for the CONV and FLASH data, and one with common α and β parameters for all data. The residuals were tested for normality using the Kolmogorov-Smirnov test, and the F-test was used to determine whether the fit was significantly improved by using separate parameters. Using the model fitted with separate α and β parameters for the CONV and FLASH data, dose-modifying factors (DMF) were determined as the ratio of DFLASH/DCONV at a survival fraction of 0.1 (SF=0.1) and 0.01 (SF=0.01). Boot-strapping was used to calculate the median and the interquartile range of the DMF. In addition, the difference in survival fraction at the individual dose levels were tested for statistical significance, without assuming normality, by using the Wilcoxon rank-sum test. All tests were two-sided with a chosen significance level of 5%. Experiments were repeated 2-4 times.RStudio v. 1.0.136 (RStudio Team (2015). RStudio, Inc., Boston, MA, URL ic model   show a distinct behavior compared to the other cell lines, with a larger DMF and also earlier cell cycle arrest. In comparison with HeLaearly passage, the HeLasubclone was considerably more radioresistant and lacked a shouldered survival curve for the CONV-irradiated samples. It has previously been reported that phenotype changes can occur with high passage numbers, affecting radiation responses  with two different doses (3 Gy and 6 Gy) indicated an earlier radiation induced G2/M-synchronization, suggesting a peak of the G2/M-arrest at a time point before 24 h , showed a lower number of foci for FLASH compared with CONV for normal lung fibroblasts, but no differences for the A549 lung cancer cell line . Cell cyFLASH effects are typically seen at doses ≥ 10 Gy. The current study showed a separation of the survival curves at doses below 10 Gy. Interestingly, also using a low dose of 4 Gy, Chabi et al. found FLASH irradiation to be more efficient than conventional dose rate exposures for two cases of T-cell lymphoblastic leukemia (T-ALL), but an opposite relation for a third case . The resin vivo experiments also suggest an iso-effective tumor control compared with CONV , the average dose rate varied between the different dose levels. However, the dose-per-pulse and the instantaneous/pulse dose rate were constant and the average dose rate exceeded 800 Gy/s at all dose levels. Further, we have chosen to perform a pure in vitro, and that the magnitude of the FLASH effect differed between the cell lines. The results indicate that the FLASH effect cannot be solely explained by TOD and that other mechanisms are involved. The nature of such possible biological susceptibilities and their dependence on oxygen concentrations will be subject to further investigations.To conclude, we have found a FLASH effect under normoxic conditions for several cell lines gabriel.adrian@med.lu.se.The datasets presented in this article are not readily available but the datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request. Requests to access the datasets should be directed to GA, CC, SM, and KP designed the study. GA, SB, EK, KB, MG, and KP conducted the experiments. GA, CC, EK, SM, SB, KP, and AW analyzed and interpreted the data GA, KP, and CC drafted the manuscript. Critical revision of the manuscript for important intellectual content – all authors. GA and CC did the statistical analyses. All authors contributed to the article and approved the submitted version.This work was supported by Mrs Berta Kamprad Foundation (grant no 2020-19-301), Swedish Cancer Society (grant no 20 1298), John and Augusta Persson’s Foundation and governmental research funding (ST-ALF). Funding for AW was provided by the Swedish Society for Medical Research (SSMF).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Recently, we read a paper in Frontiers in Cell and Developmental Biology “Anti-tumor Effect of Oleic Acid in Hepatocellular Carcinoma Cell Lines via Autophagy Reduction.” Oleic Acid (OA) may be a promising drug candidate for the treatment of hepatocellular carcinoma (HCC). However, some problems existing in this paper need to be commented.With great interest we read a paper “Anti-tumor Effect of Oleic Acid in Hepatocellular Carcinoma Cell Lines via Autophagy Reduction” published in Frontiers in Cell and Developmental Biology  for 48 h . However, authors demonstrated that OA significantly suppressed cell proliferation and induced cell death of HCC cells after incubation with the same concentrations (300 μM) of OA for the same time (48 h) . These results strongly suggested that the cause responsible for inhibition of migration and invasion of cells could be due to apoptosis induction of OA. Authors could treat the cells with OA at a lower concentration or for a short time when studying the inherent effects of drugs on the migration and invasion ability of cells without apoptosis induction. This made the results more convincing.JL: study concept and design, data analysis, methodology, drafting manuscript, review and editing, and supervision.The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Chronic kidney disease (CKD) and acute kidney injury (AKI) are public health problems, and their prevalence rates have increased with the aging of the population. They are associated with the presence of comorbidities, in particular diabetes mellitus and hypertension, resulting in a high financial burden for the health system. Studies have indicated Klotho as a promising therapeutic approach for these conditions. Klotho reduces inflammation, oxidative stress and fibrosis and counter-regulates the renin-angiotensin-aldosterone system. In CKD and AKI, Klotho expression is downregulated from early stages and correlates with disease progression. Therefore, the restoration of its levels, through exogenous or endogenous pathways, has renoprotective effects. An important strategy for administering Klotho is through mesenchymal stem cells (MSCs). In summary, this review comprises in vitro and in vivo studies on the therapeutic potential of Klotho for the treatment of CKD and AKI through the administration of MSCs. The Klotho gene was first introduced and described in 1997 by Kuro, et al., as an anti-aging gene. They reported a mutant Klotho-deficient animal model, which presented phenotypes similar to age-related events in humans, such as reduced lifespan, alongside vascular calcification and cardiovascular disease . Named aIn humans, Klotho is present in two distinct isoforms—anchored on the membrane protein or as a soluble protein. Membrane-anchored Klotho is a single-pass transmembrane protein, of which the large extracellular domain is composed of two repeated sequences of 440 amino acids, named K11 and K12. These sequences can be proteolytically cleaved by different enzymes, leading to cleaved Klotho—one of the soluble Klotho forms ,4,5,6,7.In adult humans, the membrane-anchored form of Klotho is expressed especially in the parathyroid glands, sinoatrial node, choroid plexus and, mainly, in distal tubules in the kidneys . SolubleConcerning its importance in embryo development, some data with animal models have indicated that Klotho is expressed since the early stages of life. Mangos, S., et al., for instance, have reported that this protein is detected in zebrafish within 24 h postfertilization (hpf) in the brain and in the pronephric ducts, which are the primitive tubules . LikewisThe levels of Klotho decrease with aging  and in tKlotho is, in this way, an essential factor to be investigated in both the physiology and pathology of renal diseases.Chronic Kidney Disease (CKD) is a multifactorial disease , definedAlthough there are tests to detect CKD, as mentioned above, there is a scarcity of biomarkers to diagnose this disease early and precisely and avoid its progression to ESKD and other complications ,38. ThusRegarding therapeutic approaches, there are non-pharmacological options for the treatment of CKD, such as weight reduction and blood pressure control. Still considering the study of Shabaka, A., et al. , though,Some of the main characteristics of CKD are chronic inflammation, hypoxia and oxidative stress, which contribute to structural and functional changes in the kidneys, resulting in glomerular, tubular and vascular injuries . Thus, tDuring the progression of CKD, proinflammatory factors—interleukin 6 (IL-6) and tumor necrosis factor (TNF) , for exaIn spite of the above description, the mechanisms relating to CKD have not yet been fully elucidated. Strong evidence, however, has pointed out the involvement of Klotho in this process, as will be addressed next.It has been observed in several studies that there is a decrease in Klotho levels (mRNA and protein)  both in In regard to this topic, studies conducted with rodents have strongly suggested that the administration of soluble Klotho is a safe approach ,46, althThus, Klotho deficiency is not only a biomarker for CKD, but also a pathogenic factor in the development, progression and complication of this disorder . ImportaAnchored on the membrane form of Klotho is a co-receptor for fibroblast growth-factor 23 (FGF-23), a hormone that is produced by cells residing in bone, namely osteocytes, to target a distant organ, the kidney. The Klotho/FGF-23 complex is responsible for, among other biological responses, the activation of extracellular signal-regulated kinase (ERK)1/2 and serum/glucocorticoid-regulated kinase (SGK)1. These signaling pathways downregulate the expression of the main sodium phosphate cotransporter in proximal tubules, NaPi-2a, on the membrane of tubular cells, resulting in phosphaturia, that is, renal phosphate excretion in urine . ExperimConcerning other minerals, some studies propose that, in distal tubules, Klotho/FGF-23 complexes are responsible for the modulation of calcium and sodium reabsorption, through the activation of ERK1/2, SGK1 and with-no-lysine kinase 4 (WNK4) signaling cascades. These results show that, in the context of low levels of Klotho, FGF-23 might be one of the explanations for CVD risks in patients with CKD .Additionally, it is a point of interest that soluble Klotho regulates several processes, including anti-oxidation, anti-senescence, Wnt signal transduction and the anti-renin-angiotensin system (RAAS) . It alsoIn summary, these findings support the relevance of Klotho/FGF-23 in CKD progression, as indicated in Populational studies have pointed out that, in CKD patients, the increase in FGF-23 and PTH, accompanied by the decrease in 1,25 dihydroxyvitamin D3, anticipate hyperphosphatemia , which iData in the literature indicate that Klotho modulates PTH synthesis and release directly, and also through the regulation of the active form of Vitamin D and FGF-23 in plasma . In addi2+ levels, which would bring this transporter to the cell membrane and trigger the release of PTH, due to the change in the electrochemical gradient, although the exact signaling pathway involved in this model has not yet been completely elucidated [Other models have been suggested to explain the relationship between Klotho and PTH. Imura, et al., for instance, proposed binding between Klotho and Na/K-ATPase in low Caucidated .A high level of FGF-23 is observed in patients and animal models with CKD , alongsiThe levels of FGFR and Klotho, however, may differ among experimental models and stages of CKD, accompanied by differences in levels of calcium in the blood . This faRazzaque, et al., demonstrated that FGF23-deficient mice develop hyperphosphatemia and a phenotype similar to aging and to Klotho-deficient mice , includiIn short, studies have shown that both Klotho and FGF-23  are ableThe instability in mineral metabolism, as described above, is a hallmark and also an initiator of the development of mineral bone disease in CKD , which cCardiovascular disease is an important and frequent morbidity in patients with renal illness and it is related to the mortality seen in these persons .Preclinical and clinical studies have pointed out the relevance of Klotho, and also FGF-23, for the cardiovascular system and how it can be related to CVD in CKD individuals and mortality among the elderly and in hemodialysis patients ,84. The In addition to the previously mentioned results, Faul, C., et al. evaluated the relation between FGF-23 and the pathogenesis of left ventricular hypertrophy (LVH) in humans, in patients from both the Chronic Renal Insufficiency Cohort (CRIC) study—a prospective cohort study conducted with CKD individuals —and studA similar result was observed in a study conducted with nephrectomy CKD- induced rats, in which FGF-23 caused the hypertrophic growth of myocytes in vitro and induced LVH in mice, through the FGFR pathway .In addition to the previously mentioned data, Klotho deficiency is also associated with vascular calcification (VC) in CKD, although the pathogenesis of this clinical outcome is not completely understood yet. It has been reported in a preclinical study involving mice and human aorta samples, for example, that the increase of Klotho in vitro led to the inhibition of VC . FurtherIt is also interesting to mention that, as discussed in Moreover, angiotensin II (Ang II) is also a special point of interest, because of its direct association with CVD, a possible complication in chronic kidney disease, and due to its pathological role, through several different mechanisms, as well as in CKD, as will be discussed. Reports have shown that Ang II and aldosterone suppress the expression of Klotho in the kidneys and in kidney cell lines ,38. MoreThe exact mechanisms by which Ang II reduces Klotho expression or contributes to kidney fibrosis are not completely understood yet, but experiments with free-radical scavenging have indicated the inhibition of Klotho expression by both Ang II and oxidative stress, which suggests that oxidative stress is one of these mechanisms. Furthermore, in a mouse model, inhibition of 1,25-(OH)2VD3 synthesis is correlated with increased renin expression, whereas its injection suppresses renin. Furthermore, a study conducted on vitamin D receptor (VDR)-null mice showed that they expressed higher levels of renin and Ang II. The same study has proved that, in mouse kidney and in HEK 293 cells, active vitamin D3 upregulates Klotho expression, reversing the decrease in Klotho’s mRNA by aldosterone. This demonstrates that supplementation with active vitamin D3 might have a positive effect for the upregulation of Klotho expression. Taken together, these results indicate that 1,25-(OH)2VD3 is a negative regulator of RAAS .Additionally, Miao, J., et al., 2021 illustrated in mice a reverse in both mitochondrial loss of mass and the production of reactive oxidative stress with Klotho administration. Correspondingly, it is known that RAAS activation can lead to mitochondrial damage . The inhA current challenge in regard to RAAS inhibition is that the synthesis of Ang II is not affected by RAAS inhibitors (angiotensin-converting enzyme (ACE) inhibitors or Angiotensin II receptor type I (AT1) receptor blocker, for example) and there is also a stimulation of renin secretion as a consequence ,101. It As previously mentioned, CKD is a disease characterized by persistent inflammatory responses and cellular senescence . The higIndeed, studies have shown that NF-κB, one of the main proinflammatory molecules in CKD, downregulates the expression of Klotho in mouse models, and Klotho negatively modulates NF-κB, reducing the expression of this factor and the inflammatory state in CKD . These rFurthermore, through NF-κB inhibition and consequent suppression of its target genes by Klotho ,105, thiAnother proinflammatory component involved with Klotho is TNF-related weak inducer of apoptosis (TWEAK), a soluble molecule that belongs to the family of TNF cytokines and is derived from its membrane-receptor enzymatic cleavage . In vitrLastly, in addition to this information, studies with cultured rat aorta smooth muscle cells have indicated that Klotho transfection in cells resulted in a decline in NADPH-oxidase (Nox) 2 protein levels and a consequent reduction in oxidative stress in this in vitro model . This apIn summary, Klotho might be able to reduce the inflammatory state in CKD through the regulation of some inflammatory factors, such as NF-κB, and the expression of adhesion molecules as well as that of Nox2. Some of these factors, in turn, are also able to downregulate Klotho expression, as seen in the case of NF-κB and TWEAK. Thus, it is believed that the inflammatory state can also reduce Klotho levels, which could then result in negative impacts for the tissue, considering the potential anti-inflammatory activities that Klotho might have.Another relevant contributor to inflammation and the progression of CKD, and particularly to renal fibrosis, is TGF-β. Studies have shown both an upregulation of TGF-β and a downregulation of Klotho, followed by fibrosis, in rodent models with unilateral uretral obstruction (UUO), a model for CKD development . It is aAcute kidney injury (AKI) is a disease with a sudden onset , marked The worldwide prevalence of AKI is increasing and prior data in the literature have shown, for instance, that individuals who survive AKI can have a 28% rate of mortality in the first year after the onset of the disease , as wellThis syndrome has diverse etiologies, such as other prior conditions, such as sepsis, acute or chronic illnesses , ischemiThe standard diagnosis for AKI includes an increase in either serum creatinine of 0.3 mg/dL or more within forty-eight hours, in serum creatinine by 1.5 times within seven days or a urine volume inferior to 0.5 mL/kg/h for six hours ,137. A rThere are a variety of pathological processes associated with AKI, although the exact mechanisms involved in its physiopathology are not yet completely understood ,140. ProAs previously mentioned, severe AKI is considered an independent and important risk factor for the course of CKD ,155,156;In general, the renal impairment caused by this disease is reversible, although long-term outcomes might exist, as previously discussed . In addiIn this context, Klotho has been suggested to be possibly related to AKI, as will be addressed next.Studies have demonstrated that Klotho production is reduced in different models of AKI, such as in cisplatin-induced AKI  and AKI In order to evaluate the role of Klotho in AKI, the same research group induced different levels of this protein in mice. A higher resistance to injury was found in rodents with a higher expression of Klotho; hence, these animals displayed fewer kidney alterations, which indicates that the overexpression of Klotho might mitigate AKI, whereas its deficiency accentuates the disease . ConcernTaken together, these results provide evidence that there is indeed a deficiency of Klotho in AKI and that this contributes to renal damage. Moreover, they also highlight that this protein is both an early biomarker ,46 and aThe exact mechanisms through which Klotho influences AKI and is downregulated are not well elucidated yet, though; some of these will be described below. It is important to note that there are several different models for the study of AKI; cisplatin-induced renal injury is a widely accepted one  and Klot2O2 as an insult similar to IRI in rodents have highlighted that co-incubation of cells with Klotho reduced the release of lactate dehydrogenase (LDH) [During the development and progression of AKI, there is a dysregulation in cellular processes; some of them are related to Klotho. Data have indicated that the downregulation of Klotho in AKI is associated with cellular senescence and, importantly, that this process might be induced as a response to oxidative stress , which ise (LDH) . It has se (LDH) .Furthermore, Bi, F., et al., 2018, observed that Klotho is able to suppress lipopolysaccharide (LPS) AKI through the degradation—via deglycosilation—of toll-like receptor (TLR) 4 .Regarding NF-κB in studies conducted in mice, TWEAK and TNF-α were responsible for the downregulation of Klotho, and it has been reported that the mechanism involves NF-κB . FurtherHence, Klotho is seen as a potential anti-inflammatory molecule in AKI ,170,173,In addition to the inflammatory aspects mentioned above, there are also non-inflammatory events associated with Klotho and AKI.Concerning its anti-fibrotic function, Klotho is an endogenous Wnt antagonist, blocking, as a result, the activation of β-catenin. Through the inhibition of this cascade, as previously mentioned, by binding to Wnt ligands (such as Wnt1 and Wnt4), increased levels of Klotho can reduce fibrosis in kidneys and ameliorate renal function ,175. In Moreover, studies have indicated that there is cycle arrest in AKI . ResearcThere is also evidence of increased Wnt signaling pathway activation in animal models that are deficient in Klotho. This event is associated, in vitro and in vivo, with cellular senescence . Thus, KIn mouse models for AKI induced through IRI, in turn, studies have demonstrated that the delivery of Klotho leads to improvements in apoptosis, histological damage and creatinine values. It has been reported that there is an increase in HSP (heat shock protein) 7o expression according to Klotho levels in this animal model, which contributes to the amelioration of apoptosis . Hence, Moreover, autophagy is activated in several models of AKI, such as in unilateral ureteral obstruction ,185, IRI2O2. It can inferred by these results that autophagy is one of the processes through which Klotho induces protection in renal cells, since its activity is upregulated by this enzyme [Experiments with transgenic mice overexpressing Klotho have shown a positive correlation between higher autophagic flux and higher Klotho levels; at the same time, renal cells can become more vulnerable to oxidative stress when autophagy is suppressed. Furthermore, in cell culture experiments, autophagy inhibitors resulted in a decrease of Klotho’s induction of autophagy and cytoprotective effects against Hs enzyme .Further analysis has also shown that autophagy contributes to the maintenance of collagen balance in renal cells, according to experiments involving autophagy inducers and inhibitors to evaluate the expression of collagen type I (Col I) in OK cells. Likewise, transfection of OK cells with Klotho resulted in a decrease in Col I accumulation, both extracellular and intracellular. As this effect was abolished in part with the use of autophagy inhibitors, it has been suggested that this regulation of Col I promoted by Klotho might depend on autophagy flux .Moreover, there is evidence that due to the collagen degradation stimulated by autophagy ,204,205 Taken together, these results suggest that autophagy is one of the mechanisms through which Klotho protects the kidneys.The reestablishment of Klotho provides benefits concerning the progression of renal disease and other events associated with it, as it has been discussed in this review and as briefly shown in Given the importance of Klotho and its deficiency in both CKD and AKI, some studies have been conducted into strategies for elevating its levels as a therapeutic approach. Buchanan, S., et al. have reviewed studies concerning some possible interventions to increase the levels of Klotho ; some ofConcerning the approaches described in (a).DNA methyltransferase inhibitor: In regard to the DNA methyltransferase inhibitor azacytidine, it has been observed that Klotho’s promoter is located in a region rich in cytosine and guanine—a CpG island ,207 that(b).Agonists for PPAR-γ: Moving on to troglitazone and ciglitazone, these drugs are classified as thiazolidinediones and they act as agonists of peroxisome proliferator-activated receptor gamma (PPAR-γ) . One stu(c).Histone deacetylase inhibitors: The other approach discussed in (d).RAAS inhibitors: Regarding the use of RAAS inhibitors, in turn, it has been demonstrated in a rodent model of chronic nephropathy that the inhibition of angiotensin II type 1 receptor with the use of losartan results in an augmentation in Klotho expression, along with a reduction in kidney histological damage . A meta-(e).Paricalcitol: As illustrated in (f).Intermedin: In regard to intermedin, a study with rats with CKD has shown that the decrease in Klotho protein levels was overturned by intermedin in the kidneys, plasma and calcified aorta . This co(g).Statins: Statins have also been proposed as a promising approach to promote Klotho overexpression. Studies with rodents demonstrated that these drugs led both to an increase in renal Klotho levels and to the attenuation of the reduction of Klotho in nephropathy . Further(h).Recombinant protein: The administration of recombinant Klotho, in turn, is a potential strategy to increase its levels . Studies(i).Adenoviral delivery: Lastly, as described in (j).Mesenchymal stem cells and extracellular vesicles: Taking into account all the approaches discussed previously, we decided to explore the potential of MSCs due to their applicability in regenerative medicine. Currently, these are the most studied type of stem cells  and theyTaken together, these data reveal that Klotho can be used both as a biomarker and, potentially, as a therapeutic tool for renal diseases .Mesenchymal or stromal stem cells (MSCs) are cells found ubiquitously in the perivascular region. As we have previously discussed, these cells were first described nearly 50 years ago and extensive research has documented their potential to differentiate into the three germ layers in vitro . DespiteThe MSC-derived secretome comprises extracellular vesicles (EVs), such as microvesicles and exosomes that differ in their size (50–1000 nm vs. 30–150 nm), origin  and cargo  . EVs plaThe MSC-derived secretome represents is a cell-free alternative for treating kidney diseases, which can bypass some issues related to autologous and allogeneic MSCs . This seMoreover, EVs isolated from MSCs are promising strategies in kidney diseases, as they modulate several biological processes, such as oxidative stress, apoptosis, inflammation, fibrosis, angiogenesis, cell cycle, regeneration, autophagy and cell proliferation ,236.Strategies for targeting EVs to particular target cells can also be achieved through genetic modification. Therefore, these EVs can also be engineered and loaded with Klotho recombinant protein or other cargo for kidney repair, as previously described for urine-derived EVs . ImportaOther genetic modifications of EVs comprise the modification of these EVs to express targeting moieties fused with exosome-native membrane proteins, such as lysosomal-associated membrane protein 2 (Lamp2b), tetraspanins, glycosylphosphatidylinositol (GPI) and lactadherin C1C2, as well as engineering exosome-liposome hybrids ,240.Taken together, these findings shed light on the use of engineered EVs from MSCs for Klotho delivery in acute and chronic kidney diseases.However, EV-based therapy imposes some challenges, which include the recovery of EVs, purity, specificity, sample volume, efficiency, complexity, the functionality of EVs, time, cost, the need for advanced equipment and scalability . For larFurthermore, in order to standardize the characterization of MSCs and reduce their heterogeneity, the updated version of the ISCT guidelines (2016) recommended the following analyses of the properties of MSCs before they are administered for therapeutic purposes: (a) self-renewal capacity and clonogenicity assessed via the colony-forming unit fibroblast assay (CFU-f), which means that each colony arises from a single precursor cell, and the doubling-time; (b) analytical methods for measuring potency, including extensive product characterization data evaluating immunochemical (quantitative flow cytometry or enzyme-linked immunosorbent assay), biochemical (protein binding or enzymatic reactions) and/or molecular  attributes of the product; (c) functionality assays focusing on the specific therapeutic properties that are sought in vivo, in particular the angiogenic potential and the immunomodulation assays performed with MSCs and the lymphomyeloid cell population; (d) assessment of immune plasticity at both phenotypic and functional levels; and (e) karyotype . When thAdditionally, the ISCT recommends including appropriate reference materials, standards and/or controls and establishing and documenting the accuracy, sensitivity, specificity and reproducibility of the test methods used through validation on a regular basis in facilities with Good Manufacturing Practices (GMP) .However, there are some challenges when clinical outcomes are compared across studies, such as the MSC source, which most frequently includes bone marrow, adipose tissue and the umbilical cord; the route of administration, which most often includes intravenous, followed by intra-articular, intracardiac or intrathecal depending on joint, cardiovascular or neurological diseases, respectively; the number and/or frequency of cell administration; and the homing capacity and severity of the patient’s condition ,246.+CD25+FoxP3+ T regulatory cells and may explain the immunomodulatory properties of MSCs and their involvement in tissue regeneration.In a kidney disease setting, MSC efficacy is challenged by several aspects. Therefore, the inflammatory milieu in the course of kidney disease can be modified and the MSC phenotype is modulated accordingly ,248. WheAdditionally, inflammation may also affect the interaction of MSCs and monocytes. When MSCs acquire an immunosuppressive phenotype in the presence of IL-6, they produce high levels of IDO and PGE2, which contributes to polarization from monocytes (M0) to a macrophage anti-inflammatory phenotype . Conversely, the proinflammatory MSC-induced phenotype may lead to polarization from M0 to proinflammatory macrophages  ,248.Therefore, the clinical condition of the recipient also plays a key role in our understanding of the mechanisms of MSC-mediated tissue regeneration. Thus, in individuals with diabetic ketoacidosis, stem cell therapy lacks efficiency in controlling hyperglycemia . The burIn line with these findings, MSCs obtained from individuals with cardiovascular disease (CVD), the leading cause of death in CKD, show increased senescence and reduced proliferation and regenerative potential, despite studies presenting conflicting results . HoweverIn terms of alloimmune reactions in individuals receiving allogeneic MSCs, it was documented that these responses were very low ,255.Moreover, the route of MSC delivery plays a crucial role in MSC efficiency. As previously reviewed, the intravenous route is chosen more frequently for treating kidney diseases due to the relative ease of the procedure, even though MSCs are initially trapped inside the pulmonary vasculature. Intra-arterial routes, including intra-aorta, intra-renal artery and intracarotid routes, are associated with a more robust repair of kidney injury. Although the intraparenchymal route, e.g., under the renal capsule, leads to kidney improvement, this route is less practical for clinical applications .The cell source, for instance, is a great subject of debate nowadays. Bone marrow is the most used MSC source, followed by the adipose tissue and the umbilical cord . The MSCIt is not only MSC characteristics that are involved in kidney repair. The evaluation of its therapeutic potential in the most appropriate pre-clinical model that mimics renal disease in humans is also of paramount importance for advancing our understanding of MSC-based therapy. Therefore, a large body of evidence has demonstrated the impact of this therapy in acute kidney injury, including IRI, kidney transplantation and drug-induced kidney injury , as wellOne of the most important aspects of advancing MSC-based therapy is defining the MSC source, whether they are derived from allogeneic or autologous donors. As we have previously reviewed, aging and chronic diseases, in particular diabetes mellitus and DKD, may decrease the potency of MSCs, including a decrease in their homing capacity and potential for angiogenic differentiation . These fTherefore, MSCs contribute to mitigating acute kidney injury after ischemic and toxic injury , and to These findings have important clinical and pathophysiologic implications regarding the impact of MSC-based therapy on the burden of kidney diseases. As kidney diseases encompass the dysfunction of a wide range of cells, including epithelial tubular cells from distinct compartments, interstitial cells, endothelial cells, mesangial cells and podocytes, strategies to enhance MSC potency are further warranted. Therefore, preconditioning of these cells with hypoxia, pharmacological agents, trophic factors/cytokines, physical factors/material and small molecules, as well as genetic modification and the optimization of MSC culture conditions, are currently being pursued . These sImportantly, preconditioning of MSCs in a hypoxic environment with 2–5%, which mimics the MSC environment, allows these cells to remain multipotent and have a greater proliferative and migratory capacity, and exhibit low rates of senescence ,269. NotOther strategies investigated to precondition MSCs include the use of the iron chelator deferoxamine (resulting in an increase in angiogenic factors and anti-inflammatory cytokines) ; antioxiMoreover, MSCs can also be tested as carriers of genes or genetic modifications. Due to their ability to migrate to injury sites, MSCs represent a promising strategy for the delivery of genes associated with the regeneration and repair of renal tissue, functioning as a “Trojan horse” . Thus, sMSC-based therapy is equally promising in the context of genetic modifications for treating kidney diseases, including the overexpression of sirtuin-3 , erythroStromal-derived factor-1 (SDF-1), also known as CXCL12, and its receptor, the CXCR4 axis, is a crucial key pathway in cell trafficking during kidney damage. Thus, in acute kidney injury, SDF-1 mRNA levels increase more than 2.5-fold and remain as high as ~2.0-fold after 24 h within kidney cortex tissue . That inAlthough MSC-based preconditioning treatment has not been associated with harmful effects, further studies are required to verify its effectiveness in maintaining MSC properties.Looking forward, the production of a large amount of MSCs is a challenge in the advancement of MSC-based therapy in renal diseases, as multiple infusions might be required, in particular for chronic kidney disease. Therefore, automated hollow-fiber bioreactors and microcarriers have been validated for the development of the large-scale manufacturing of MSCs, providing cells with preserved characteristics and functionality in a 3D environment when compared to the manual multilayer flask method ,283. ImpIn addition to the previously discussed strategies designed to increase Klotho levels, MSCs are a promising strategy aiming to modulate Klotho expression in CKD and AKI . MoreoveA preclinical study, for instance, has been conducted with streptozotocin-induced DKD in rats, which were further injected with adipose-tissue-derived mesenchymal stem cells (AT-MSCs). It was found that these cells resulted in a reduction of typical histological alterations in the animals treated, such as tubulointerstitial fibrosis and glomerular hypertrophy. Furthermore, the authors observed a decrease in apoptosis in renal tissue, as seen by a restoration of Bax and Bcl-2 to normal levels. Interestingly, the researchers also evaluated the levels of Klotho after the intervention proposed and reported that in AT-MSC animals, the levels of this protein were higher, whereas the expression of proteins such as Wnt1 and active β-catenin were lower. A similar result has been observed in vitro, in experiments involving the co-culturing of NRK52-E cells—rat kidney clones from epithelioid and fibroblastic cells—and AT-MSCs in high-glucose medium. In these in vitro experiments, a higher expression of Klotho was reported, alongside the inhibition of upregulated proteins by the hyper-glucose medium, such as Wnt 1 and active β-catenin, when the stem cells were used. In brief, it has been demonstrated that stem cells are able to mitigate renal damage through both the upregulation of Klotho and the inhibition of Wnt/-catenin signaling .Concerning the inhibition of the Wnt/β-catenin pathway, it has been proposed by other studies that Klotho is able to bind to ligands of this pathway, along with the fact that it is negatively related to Klotho expression ,182. In In addition to the reports mentioned, studies have shown that Klotho improves BM-MSC proliferation ,288,289 Furthermore, it has been shown that modification through overexpression of the Klotho gene increases the paracrine ability of BM-MSCs, expressing higher levels of IGF-1, VEGF and HGF . Thus, tIn short, the increases in Klotho levels in kidney diseases have been associated, in different studies, with the improvement of histological damage, such as glomerular hypertrophy and tubulointerstitial fibrosis, along with reduced apoptosis and activation of the Wnt/β-catenin pathway, which is involved in the progression of kidney illnesses. At the same time, data in the literature point out that stem cells are able to modulate processes related to the attenuation of renal injury in kidney diseases, including through the regulation of Klotho expression, through their characteristic properties , althougAs previously discussed, CKD is considered a public health problem, with a rising incidence, that usually results in ESKD and other complications. There is a paucity of biomarkers that can be used for the early diagnosis of this condition. Therapeutic approaches are also limited and CKD usually progresses in patients. Therefore, considering that CKD is a complex disease, the development of new strategies for the treatment of this illness is of pivotal importance and the use of more than one therapeutic measure might be interesting. Similarly, AKI might lead to a higher risk of CKD and ESKD development and it is associated with high mortality in patients as well, both in hospitalized individuals and in those who are not hospitalized. Its incidence is also rising; however, there is a lack of therapies available and a lack of biomarkers for the early stages of the disease. Thus, the study of new potential therapeutic approaches for both diseases remains relevant.In this regard, MSC therapy is an emerging approach for the treatment of acute and chronic kidney diseases. Due to their properties, these cells are an object of interest for many ongoing studies and clinical trials ,295. IndAs previously explored in this review, an important point of interest concerning MSC therapy is the administration of MSC-derived EVs, since most of these cells’ pro-regenerative effects occur because of their paracrine signaling. Hence, this is a promising approach for the treatment of both CKD and AKI. In addition to the interesting properties mentioned above in relation to their microRNA cargo, these vesicles exhibit a preferential tropism to the damaged kidney. Studies have demonstrated, for example, that in comparison with sham controls, in which the vesicles accumulate mainly in the liver, the damaged tissue uptakes the MSC-EVs more efficiently ,297. FurSimilarly, it has been found that the administration of MSC-EVs from human umbilical cord-derived MSCs into a mouse model of IRI reduces cell death and induces the proliferation of tubular cells . CharactFurthermore, data in the literature have shown that the downregulation of microRNA in MSC-EVs decreases the protective properties of these cells in AKI. MSC-EVs containing microRNA in its inactive form failed to promote healing in a murine glycerol model of kidney injury, whereas EVs carrying mature and active microRNA ameliorated the damage, reduced the apoptosis of epithelial tubular cells and promoted a proregenerative expression profile in the injured tissue .Interestingly, a review of studies with DKD murine and primate models showed an increase in survival and an improvement of functional and structural parameters after MSC transplantation  MSC- andStill concerning DKD patients, MSC-based clinical trials reported significant improvements in renal function, with decreased levels of serum creatinine, blood urea nitrogen (BUN), albuminuria and kidney hypertrophy . MoreoveHence, the administration of MSCs-EVs is a potential approach to MSC therapy for kidney diseases. As shown in several studies discussed here and some others that will be discussed in the next section, these vesicles present tropism for injured tissue, alongside the beneficial effects provided by their cargo.Regarding the previously discussed information, the MSC-derived secretome functions as a mechanism of cell–cell communication by which MSCs can modulate tissue repair and regeneration. This secretome is composed of a wide range of soluble factors and vesicles carrying cytokines, chemokines, growth factors and proteases, as well as messenger RNA (mRNA), microRNA(miR) and DNA . Among tSome examples of abundant microRNAs in MSC-derived EVs include miR-125a-5p, miR-26a-5p and miR-191-5p, which may have an important role in renal physiology and repair. Among these molecules, miR-125a-5p acts on the attenuation of the inflammatory response as it promotes the polarization of macrophages M2 by targeting tumor necrosis factor receptor (TRAF) 6 mRNA and regulating its expression ,307. TheMoreover, miR-26a-5p mediates the maintenance and homeostasis of glomerular cell types . It is hThus, as indicated by several studies, some miRs might be unbalanced in renal diseases. On the other hand, they are able to promote the amelioration of the inflammatory burden in the renal tissue, as well as the prevention of extracellular matrix accumulation. They are also important for the maintenance of cells’ physiology, such as that of podocytes. Therefore, among the components of MSCs-EVs, miRs present important roles in kidney repair.In conjunction with the above information, as further evidence of its emergent potential as a complementary strategy to the current treatment, a study has shown decreased sodium-glucose co-transporter-2 (SGLT2) expression on renal tubular cells after MSC administration in rhesus monkeys . SGLT2 iIn addition to the topics discussed in this review, Klotho expression is also important in the setting of CKD and AKI. As addressed here, Klotho overexpression has renoprotective effects, whereas its downregulation is associated with the progression of kidney diseases. Interestingly, individuals with kidney injury have dysregulated miRNA expression and activity that may impair Klotho expression through epigenetic and post-transcriptional mechanisms. In fact, it has been found that diabetic patients exhibit higher levels of miR-200c, which is related to endothelial dysfunction and Klotho downregulation under oxidative stress conditions ,319. In Accordingly, the transplantation of MSCs genetically modified with the Klotho gene would be a strategy to restore and promote tissue repair through different mechanisms and signaling pathways. Again, MSCs can act as therapeutic carriers for gene delivery and genetic engineering products, functioning as a “Trojan horse” . MoreoveDespite the promising results in relation to clinical applications, MSC therapy still has some challenges to overcome. The heterogeneity of pre-clinical and clinical studies and features of cell sources may hamper the establishment of a standardized protocol for the procedure ,257,295.Lastly, in order to continue advancing MSC-based therapy, the production of a large amount of these cells is a key aspect. MSCs cultured for a prolonged period may be affected by a disturbance in their cellular structure and function. Thus, chromosomal instability and aberrations of MSCs are controversial, as some researchers have documented their occurrence  and otheThe restoration of Klotho levels is a strategy designed to ameliorate the damaging renal microenvironment and gene expression profile present in both chronic and acute kidney diseases. Likewise, MSCs present great potential as allies for this therapeutic approach, due to their characteristic properties—including the release of EVs—and also because of their possibility to act as gene carriers. There are some challenges to be overcome in regard to the use of these cells in vivo, such as the influence of their potency on their biological performance, as well as their large-scale production and safety issues. To date, however, no clinical trials have reported malignant transformations of these cells. Therefore, the synergism between Klotho and MSCs might be an interesting therapeutic strategy for reducing the burden of CKD and AKI, in particular when associated with the current pharmacological and non-pharmacological approaches.


Artemisia santolinifolia ethanol extract (AS) was assessed for its chemosensitizer ability when combined with the conventional anticancer drug, docetaxel (DTX), against non-small cell lung cancer (NSCLC). SRB assay was used to determine cell viability for A549 and H23 cell lines. The potential for this combination was examined by the combination index (CI). Further cell death, analyses with Annexin V/7AAD double staining, and corresponding protein expressions were analyzed. Surprisingly, AS synergistically enhanced the cytotoxic effect of DTX by inducing apoptosis in H23 cells through the caspase-dependent pathway, whereas selectively increased necrotic cell population in A549 cells, following the decline in GPX4 level and reactive oxygen species (ROS) activation with the highest rate in the combination treatment group. Furthermore, our results highlight the chemosensitization ability of AS when combined with DTX. It was closely associated with synergistic inhibition of oncogenesis signaling molecule STAT3 in both cell lines and concurrently downregulating prosurvival protein Survivin. Conclusively, AS could enhance DTX-induced cancer cells apoptosis by abrogating substantial prosurvival proteins’ expressions and triggering two distinct cell death pathways. Our data also highlight that AS might serve as an adjunctive therapeutic option along with a conventional chemotherapeutic agent in the management of NSCLC patients.Conventional chemotherapy remains an integral part of lung cancer therapy, regardless of its toxicity and drug resistance. Consequently, the discovery of an alternative to conventional chemotherapy is critical.  It is estimated that more than two million new lung cancer cases are diagnosed in the world annually. Most of them are non-small cell lung cancer  . IncreasTraditional medicine has been used since ancient times and has gained increasing attention as a remarkable source for finding new drugs with few side effects . The benDocetaxel (DTX), an antimicrotubule taxane binding to the β-tubulin subunit of microtubulin, is a standard regimen for both first- and second-line treatment in advanced NSCLC ,12. AlthArtemisia santolinifolia is a “wormwood” semi-shrub with three-pinnatisect leaves, widely distributed in Central Asia to Eastern Europe, ranging between 400 and 500 different species  solution prepared with 0.1% formic acid in 30% ACN, was delivered from the auxiliary pump at a flow rate of 0.2 μL/min to the reference sprayer of the LockSpray source.m/z 500. All analyses were performed using positive mode ESI using a LockSpray source. The lock mass channel was sampled every 30 s. The mass spectrometer was calibrated with a Leucine enkephalin solution (200 fmol/μL) delivered through the reference sprayer of the LockSpray source. Accurate mass LC-HDMSE data were acquired in an alternating, low energy (MS) and high energy (MSE) mode of acquisition with a mass scan range from m/z 50 to 1000, using a capillary voltage of 2 kV, a source temperature of 150 °C, and a cone voltage of 30 V. The mass measured accuracy was <5 ppm when the instrument operated with the lock mass, and the accuracy was the same between low energy and high energy MS scans. The spectral acquisition time in each mode was 1.0 s with a 0.1 s inter-scan delay. In low-energy HDMS mode, data were collected at a constant collision energy of 2 eV in the trap and transfer cells. In high-energy HDMSE mode, the collision energy was ramped up from 15 to 30 eV in the transfer cell only.LC-HDMSE Data were acquired in resolution mode with UNIFI Scientific Information System. The mass spectrometer operated in resolution mode with a typical resolving power of at least 40,000 FWHM at t-test and analysis of variance (ANOVA) with pair-wise comparisons. Statistical significance was defined at * p < 0.05, ** p < 0.01, and *** p < 0.001 vs. corresponding control.All data were treated with Sigma Plot  software. The results are presented as means ±SE of three independent experiments. Differences between the two groups were calculated using Student’s