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


Breast cancer is one of the most frequently diagnosed cancers in the world and is also the leading cause of death in women diagnosed with this disease. Recently, APOBEC3 proteins have been identified as potent mutagenic agents of genomic DNA associated with the onset, progression and treatment resistance of various types of cancer. On the other hand, Vif1 from HIV-1 and Vif2 from HIV-2 are proteins encoded by HIV-1 and HIV-2, respectively, that during viral infection plays a crucial role in the inhibition/degradation of APOBEC3. In this work it was tested the hypothesis that both Vif1 and Vif2 mediated APOBEC3 inhibition will increase the cytotoxicity of docetaxel in a triple-negative breast cancer cell line.Breast cancer HCC1806 cell line was used as well as two new derived cell lines, with vif1 and vif2 genes integrated and expressing vif in fusion with Zs Green fluorescent protein, mentioned hereinafter simply as VIF-1 and VIF-2 cells. Cell viability assays were performed by MTT reduction after 24 h and 48 h exposure of cells  to different concentrations of docetaxel.p-value > .05 – independent sample t-test). Additionally, after 48 h exposure to docetaxel, cell survival also decreases significantly to values between 38 and 43%  at any of the docetaxel concentrations tested ( and 43% , but aga


Bladder cancer (BC) in dogs is often lethal at the time of diagnosis. Therefore, there is a constant need for novel research on improvements of its characterization and treatment. Due to high cost and limited number of available dog patients, in vitro models of canine BC have been increasingly used for the last 25 years. In the present article, we present existing in vitro models of canine BC, including available simple  and more complex  models.Bladder cancer (BC) constitutes approximately 2% of all spontaneously occurring cancers in dogs. It is characterized by a devastating clinical course in most cases, which emphasizes a constant need for the development of novel methods of disease characterization and treatment. Over the past years, advances in cell engineering have resulted in the development of various canine in vitro models of BC, emerging as complements for in vivo research. In this article, we aimed to review the available data on existing in vitro models of canine BC, focusing primarily on their characteristics, applications in veterinary medicine, as well as advantages and disadvantages. The most commonly used in vitro models of canine BC comprise immortalized cell lines grown as adherent monolayers. They provide an unlimited supply of research material, however, they do not faithfully reflect the conditions prevailing in vivo, since the spatial cellular interactions are lost. The importance of the three-dimensional (3D) features of solid tumors in relation to carcinogenesis or drug response process has resulted in the development of the first canine 3D models of BC available for in vitro research. So far, results obtained with in vitro and in vivo research should be interpreted together. With the constantly growing complexity of in vitro models of BC cancer, animal-based research might be reduced in the future. Bladder cancer (BC) constitutes approximately 2% of all spontaneously occurring cancers in dogs . With esThough obvious benefits that could be gained from the research of canine BC in vivo, there are some inevitable limitations , making extensive in vivo research difficult ,2. CurreIn the present article, we aimed to review the available data on existing in vitro models of canine BC as a tool in veterinary research, focusing primarily on their characteristics, potential applications in veterinary medicine, and advantages and disadvantages. To the best of our knowledge, this is the first review summarizing the current evidence regarding this topic. We conducted a literature search using two electronic databases, namely, Pubmed and Scopus. The most recent search was performed on 28 January 2022. Screening of the literature was conducted using the following search string:  AND (“cancer” OR “carcinoma” OR “neoplasm”) AND (“in vitro” OR “model” OR “cell*” OR “culture”) AND (“dog” OR “canine”). Auto-alerts in Medline were run, as well as reference lists of original articles and review articles for further eligible data. We exclusively included data regarding in vitro models of canine bladder TCC. Only papers in English were considered eligible without restrictions on publication year. The flow diagram of the study selection process is presented in The most commonly used two dimensional (2D) models of canine BC comprise immortalized cell lines and primary cell cultures grown as adherent monolayers in appropriate media. The first immortalized canine BC cell line, called K9TCC, was established by Knapp et al. in 1995 . Since tThe majority of canine BC cell lines express specific cancer-related markers, resembling those presented by the primary tumors in vivo. Due to differences in the development process (mainly related to primary tumor characteristics), the available canine TCC cell lines differ in terms of expressed biomarkers . This alTo date, only a few studies have provided data regarding high-throughput molecular characterization of existing canine BC cell lines. Initial molecular characterization (including genotypic data) of several canine BC cell lines was performed by the Flint Animal Cancer Center (FACC) . SubsequIn vitro studies using BC cell lines play a significant role in the novel drug discovery and development process, providing crucial data on drug effects in the early preclinical stages. Such information is of paramount importance in the decision-making process for drugs moving forward into more expensive and time-consuming in vivo clinical trials. Initial studies using canine TCC cell lines were extensively focused on non-selective cyclooxygenase inhibitors  and various chemotherapeutic agents . FollowiCanine BC cell lines can be also used for investigation of other forms of anti-cancer therapy. As an example, Parfitt et al. investigated and characterized the radiosensitivity and capacity for cellular damage repair of canine BC cell lines . AuthorsConventional 2D cultures have several advantages supporting their important role in preclinical research. Research using cell lines is significantly less expensive than in vivo animal studies and provide an unlimited supply of material, which is widely available and easy to propagate under completely controlled and reproducible environmental conditions . NeverthThe importance of the three-dimensional (3D) features of solid tumors in relation to carcinogenesis or drug response process has prompted efforts to develop in vitro models mimicking in vivo tumor growth more precisely. Examples of 3D culture systems include multicellular aggregates grown as spheroids, scaffold-based models grown within polymer networks, and organoids defined as stem cell-containing self-organizing structures possessing multiple features of the original tumor ,32. DespThe first 3D models of canine BC were established by Elbadawy et al. . They ge3D cell culture approaches hold great potential and offer complex systems for various purposes, such as disease modeling and investigation of anticancer drug efficacy. The similarities in the drug responsiveness among the 3D in vitro models and the in vivo models might largely be due to their similarities in enhanced cellular interactions via adhesion and secretion of soluble factors of tumors ,36. ThesAlthough significant advances have been made over the past years, modeling the complexity of canine BC in in vitro models has not been completely successful. There are still various challenges, including the need for extensive molecular characterization of existing cell lines or creation of new reliable 3D models, incorporating multicellular cultures and diverse cellular microenvironments. So far, results obtained by in vitro and in vivo research should be interpreted together. With the constantly growing complexity of in vitro models of BC cancer, animal-based research might be reduced in the future


Eupatorium japonicum Thunb. of the plant family Asteraceae is a popular traditional herb in Vietnam. However, its chemical constituents as well as bioactive principles have not been investigated yet. We investigated the phytochemistry of E. japonicum in Vietnam and isolated seventeen compounds (1–17) including phytosterols, terpenoids, phenolic acids, flavonoids, fatty alcohols, and fatty acids. They were structurally determined by MS and NMR analysis. Except for compounds 6 and 12, all the other compounds were identified for the first time from E. japonicum. Since many sesquiterpene lactones with α-methylene γ-lactone ring are reported as anti-inflammatory and anticancer agents, eupatoriopicrin (10), 1-hydroxy-8-eudesma-4(15),11(13)-dien-6,12-olide (11) were selected among the isolates for biological assays. Compound 10 was identified as the main bioactive sesquiterpene lactone of E. japonicum showing its potent anti-inflammatory and cytotoxic activity through inhibiting NO production and the growth of HepG2 and MCF-7 human cancer cell lines. For the first time, eupatoriopicrin (10) was demonstrated to strongly inhibit NTERA-2 human cancer stem cell (CSC) line in vitro. It is noticeable that the cytotoxicity of eupatoriopicrin against NTERA-2 cells is mediated by its apoptosis-inducing capability of 10 as demonstrated by the results of Hoechst 33342 staining, flow cytometry apoptosis analysis, and caspase-3 activity assays. The biological activities of the main bioactive constituents 1–7, 10, 12, and 15 supported the reported anti-inflammatory and anticancer properties of extracts from E. japonicum. Eupatorium species used in Vietnamese traditional medicine such as E. fortunei -prop-2-en-oic acid (13) [p-menth-1-ene-3,6-diol (14) [O-rutinoside (rutin) (15) [O-methyl ether (16) [17) [1H-NMR, and 13C-NMR) with reported literature values. Compounds 1–5, 7–11, and 13–17 were isolated for the first time from E. japonicum (The MeOH extract from the leaves of tate (1) , ß-amyritate 2) , α-amyriyrin 3) , ß-amyriyrin (4) , ß-sitoserol (5) , stigmas (5) [6) , β-sitos , α-amyr , ß-amyr (7) [8) , stigmasside (9) , eupator(9) [10) , 28, 1-h10) [11) , 30, caf11) [12) , (2E)-3-iol (14) , quercetin) (15) , quercether (16) , and kae16) [17)  which weaponicum .10 and 11 is shown in 10 strongly inhibited NO production in LPS-stimulated RAW 264.7 cells in a dose-dependent manner with an IC50 value of 7.53 ± 0.28 µg/mL. The positive control L-NMMA exhibited its IC50 value of 8.21 ± 0.84 µg/mL. Cytotoxicity of 10 was observed at the active concentrations; the cell viability was 43.27% at 4 µg/mL (NO inhibition: 93.83%), but up to 85.64% at 0.8 µg/mL (NO inhibition: 85.02%) (10 was reported to show strong anti-inflammatory activity through the inhibition of IL-8 and TNF-α release in lipopolysaccharide- (LPS-) stimulated human neutrophils [10 as a lead compound towards inflammation. Compound 11 did not display potent suppression on NO production at all concentrations tested . The highest inhibition of NO production was 48.02% at 100 µg/mL with the cell viability reaching 95.25%. Among the isolates 1–17, the inhibition of NO generation, inflammatory cytokines, tumor necrosis factor-α, or COX-2 enzyme was reported for triterpenes 1–4 [5–7 [12 and 15 [Nitric oxide (NO) participates in various responses of host resistance to various pathogens and joins to adjust various aspects of life such as vascular integrity, maintains hemostasis, or modulates neural activity , 37. The 85.02%) . Therefotrophils . Altogetenes 1–4 –42, phyt2 and 15 , 45. Thein vitro and in discovering novel anticancer compounds against CSCs, a research area of great interest nowadays. The cytotoxicity of compounds 10 and 11 on human breast cancer cell line (MCF7), human hepatocellular carcinoma (HepG2), and pluripotent human embryonal carcinoma cell line (NTERA-2) was accessed using SRB assay. As shown in 10) showed strong cytotoxic activity on all tested cell lines with IC50 values of 1.22 ± 0.10 µg/mL, 0.94 ± 0.12 µg/mL, and 0.88 ± 0.05 µg/mL, respectively. Compound 11 did not show cytotoxic effects on HepG2, MCF-7, and NTERA-2 cell lines at the concentrations tested. Hitherto compound 10 expressed its cytotoxic activity against KB, HeLa, HL-60, EN19, EAT, P388, FIO 26, and L5178Y cell lines [10 may be associated with its α-methylene-γ-butyrolactone moiety [in vivo study showing the tumor growth delay after administration of eupatoriopicrin [10 against human CSCs (NTERA-2) as well as human cancer cell lines HepG2 and MCF-7. Glutathione (GSH) depletion is a central signaling event that regulates the activation of cell death pathways [in vitro study by Woerdenbag et al. showed that significant GSH reduction began to occur with concentrations of eupatoriopicrin ≥1 μg/mL in FIO 26 cells [10, we assayed further the apoptosis-inducing activity of 10 in NTERA-2 cells.NTERA-2 is an embryonal carcinoma cell line, which expresses a gene profile related to pluripotency of stem cells such as OCT3/4 and SSEA-4. The cell line presents a lot of early embryo antigens that could be used to identify undifferentiated human embryonic stem cell (hESC) , 47. In ll lines , 48, 49.iopicrin . The prepathways . An in v26 cells . Eupator10 on NTERA-2 CSC line, 10 was further evaluated for its apoptosis-inducing activity to better understand the possible apoptosis pathway. We examined the possible induction of apoptotic cell death by 10 using flow cytometry analysis and activation of caspase-3 in CSCs. Thus, NTERA-2 cancer cells were treated with 10 and stained with Hoechst 33342. Using this nuclear staining dye, apoptotic cells with condensed and fragmented nuclei were detected, while most negative control cells showed nuclei with homogeneously staining morphology  activation (Franco and Cidlowski). Caspase-3 is one of the key enzymes in apoptosis and is activated in both the intracellular and extracellular apoptotic pathways [10 significantly induced caspase-3 production at all tested concentrations (p < 0.05) . Higher or lower concentrations also mediated caspase-3 production but the induction was not as strong as that at 1.0 µg/mL. These results indicated that this concentration might be the optimized concentration of 10 to induce apoptosis in the NTERA-2 CSC line through the activation of caspace-3.In order to confirm the apoptosis-inducing activity of compound pathways , 55. Thipathways . Compoun < 0.05) . Thus, c10 expressed the potential activity against CSCs by apoptosis and/or necrosis induction. A challenge of cancer therapy is multidrug resistant, which is closely related to CSCs [10 that reduce survival rates of CSCs and induce apoptosis attract considerable attention.Throughout,  to CSCs . Multidr to CSCs –60. TherE. japonicum. Belonging to the privileged group of anti-inflammatory and anticancer structures, two sesquiterpene lactones 10 and 11 were selected for biological assays. Eupatoriopicrin (10) was found to strongly inhibit NO production, MCF-7, HepG2, and NTERA-2 cancer cell lines; meanwhile, compound 11 was not active. The cytotoxicity on the NTERA-2 cancer cell line is related to significant apoptosis induction by the action of 10 in the concentration- and caspase-dependent manners. The importance of the findings is that the study provides a chemical and biological basis for developing new approaches to explore therapeutic values of extracts or herbal medicines containing the herb E. japonicum. In addition, anti-inflammatory and anticancer botanical drugs enriched with bioactive constituents can be formulated and prepared from E. japonicum. Eupatoriopicrin (10) is also exposed in the study as a lead compound targeting inflammation or cancer diseases. Further projects on the synthesis of chemical derivatives or analogs of 10 addressing optimization of drug safety and effectiveness in combating inflammatory or cancer diseases are underway.In this study, 15 compounds out of 17 isolates were isolated for the first time from


TET2 mRNA and miR-92a-3p and miR-92a-5p mature miRNAs in NHL cell lines, tumour samples, and the whole blood gDNA of an NHL case control cohort. Increased expression of both miR-92a-3p and miR-92a-5p and aberrant expression of TET2 was observed in NHL cell lines and tumour tissues, as well as disparate levels of dysfunctional promoter CGI methylation. Both miR-92a and TET2 may play a concerted role in NHL malignancy and disease pathogenesis.MicroRNAs (miRNAs) are well known for their ability to regulate the expression of specific target genes through degradation or inhibition of translation of the target mRNA. In various cancers, miRNAs regulate gene expression by altering the epigenetic status of candidate genes that are implicated in various difficult to treat haematological malignancies such as non-Hodgkin lymphoma by acting as either oncogenes or tumour suppressor genes. Cellular and circulating miRNA biomarkers could also be directly utilised as disease markers for diagnosis and monitoring of non-Hodgkin lymphoma (NHL); however, the role of DNA methylation in miRNA expression regulation in NHL requires further scientific inquiry. In this study, we investigated the methylation levels of CpGs in CpG islands spanning the promoter regions of the miR-17–92 cluster host gene and the TET2 gene and correlated them with the expression levels of  Non-Hodgkin Lymphoma (NHL) is a class of cancer that originates in the lymphatic system, caused by an over-proliferation of malignant B-cells. NHL is one of the most common cancers in the United States, and lymphoma is the fifth most common cancer in Australasia , therefoUnderstanding the cell of origin also plays a significant role in understanding NHL classification, progression, and prognosis . B-cell Dysregulation of miRNAs is a hallmark of both cancer initiation and metastasis, through the regulation of gene expression via post-transcriptional repression and mRNA degradation . It is wTET2 gene function in bone marrow cells leads to increased immature B-cells resulting in lymphomagenesis  differentiation, balanced B-cell terminal differentiation, and oxidative demethylation , with tumour tissue from 11 NHL cases  with a confirmed diagnosis of NHL (5 DLBCL and 6 FL) . A cohor2 incubator. Cell count and viability were assessed using Trypan Blue exclusion method in a 1:1 ratio of cell suspension to dye and measured on a TC10™ automated cell counter. Cells were split into a 1:2 ratio, with fresh media was added every second day until >80% confluence and 90% viability were achieved.Studies were conducted using four commercial immortalised cell lines: Raji (BL), Toledo (DLBCL), SUDLH4 (DLBCL), and Mino (Mantle cell lymphoma (MCL)). Vials were stored in liquid nitrogen and rapidly thawed and seeded in a T-75 culture flask in 25 ml RPMI-1640, supplemented with 10% FBS and 1% Penicillin-Streptomycin antibiotics. The cells were incubated at 37°C in a humidified 5% COMIR17HG) promoter region and the miR-92a to TET2 promoter was performed using MethHC 2.0 software.Computational analysis of miRNA and target gene prediction was performed using miRbase , miRTarB® SV Genomic DNA Purification System (Promega). DNA quality and quantity were quantified using a Nanodrop spectrophotometer, determined by A260/A280 ratio .gDNA was previously extracted from whole blood, collected into EDTA tubes, using an in-house salting-out method as evaluated by Chacon et al. . Both NHBisulfite conversion of 500 ng of NHL B-cell line and control PBMC DNA and 750 ng of DNA from each patient/control DNA sample and NHL tumour sample was performed using the EZ DNA Methylation™ Kit (Zymo Research) according to the manufacturer’s protocol, with a modification to the elution volume, from 10 to 40 µL.® Reagent (ThermoFisher) and the Direct-zol RNA MiniPrep Kit (Zymo Research) from cell lines, control PBMCs, and NHL tumour samples. Total RNA, including miRNA, was reverse transcribed using the MiScript II RT Kit (Qiagen) for all samples.Total RNA was extracted using TRIzol® Green PCR, Qiagen) and mRNA transcripts . Customised forward and reverse primers  were designed for the miRNAs and TET2 mRNA transcripts of interest (TET2 expression was measured under the following conditions: 50°C for 2 min (x1 cycle), 95°C for 3 min (x1 cycle), 95°C for 3 s, 60°C for 30 s (x50 cycles), melt curve analysis 60–95°C. Endogenous controls were miR103 for the miRNA assays and 18 S ribosomal RNA for the TET2 assays. Specific amplification of targets was confirmed via melt curve analysis and gene expression was calculated using the relative quantification method (ΔΔCt).Analyses were performed by real-time quantitative PCR (Q-PCR) to measure miR-92a mature miRNAs  as per the manufacturer’s instructions using PyroMark Q96 Gold reagents (Qiagen). Pyrosequencing output analysis was performed using the Qseq software .The UCSC genome browser was used to identify CGI regions in the  regions . Pyromar regions . PrimersStatistical analysis of both expression and methylation data was performed in GraphPad Prism, with a Kruskal–Wallis (KW) test of significance and a post-hoc Dunn test for comparisons of individual groups.TET2 mRNA were assayed by RT-qPCR, and upstream promoter CGI methylation for each gene was assayed by bisulfite pyrosequencing, in 4 biological replicates of NHL cell lines SU-DHL-4, Mino, Raji, and Toledo, and in PBMCs of 6 healthy controls (p = 0.0145) and miR-92a-5p expression increased in Mino (p = 0.0013), Raji (p = 0.0016), and Toledo (p = 0.0200) cells compared to controls . Differential methylation was also observed between SU-DHL-4 and Raji cells (p = 0.0237). and between Mino cells and Raji (p = 0.0029) and Toledo (p = 0.0278). Toledo (p = 0.0329) cells showed increased methylation in region 2 when compared to controls  and Toledo (p = 0.0045) cells compared to controls (p = 0.0016), Raji (p = 0.0385), and Toledo (p = 0.0004) cells. Upstream promoter hypermethylation was observed in SU-DHL-4 and Raji cell lines when compared to controls. (p < 0.0001). SU-DHL-4 cells were also significantly different to Mino and Toledo cells (p < 0.0001), and Raji cells were significantly different to Mino and Toledo cells (p < 0.0001).controls . DiffereTET2 mRNA were assayed in NHL patient tumour biopsies and compared to healthy control PBMCs by RT-qPCR (p = 0.0017), and differential methylation was observed between controls and FL tumours in region 1 of the MIR17HG upstream promoter CGI (p = 0.0203).The expression of miR-92a-3p and miR-92a-5p mature miRNAs and  RT-qPCR . PromoteTET2 mRNA expression was observed in both DLBCL (p = 0.0164) and FL (p = 0.0240) patient tumour biopsies compared to controls (p = 0.0300).Increased controls , and decMIR17HG promoter CGI was observed in the NHL cohort compared to the controls (p < 0.0001). Furthermore, differential DNA methylation was observed between healthy controls and NHL patient subtypes, with increased levels of DNA methylation observed in the FL (p = 0.0004), MCL (p = 0.0046), BL (p = 0.0062), and LBL (p = 0.0004) patient groups (MIR17HG upstream promoter CGI (p = 0.0041) and MCL (p = 0.0398) subtypes when the specific diagnosis was considered (TET2 promoter CGI methylation was observed in NHL cases compared to controls (p = 0.0159), and between healthy controls and FL subtype (p = 0.0210).DNA methylation of CpGs in each region was assayed by bisulfite pyrosequencing of whole blood gDNA from an NHL case-control cohort, comprised of 80 cases with age and sex-matched controls . Increast groups . No diffoter CGI ; howevernsidered . DecreasTET2 gene were quantified by bisulfite pyrosequencing and correlated with the expression levels of TET2 mRNA and mature miR-92a-3p and miR-92a-5p miRNAs. Gene expression was assayed in NHL B-cell lines, NHL tumour samples, and healthy PBMCs. Methylation of promoter CGIs was measured in the DNA of several NHL B-cell lines, in NHL tumour samples, in healthy PBMCs, and in whole blood gDNA of a retrospective NHL case-control cohort.In the last decade, both the role of miRNAs in malignancy and their viability as biomarkers for malignancy has been a rapidly expanding area of research. The mechanisms behind miRNA expression are not fully understood; however, aberrant methylation of promoter regions of miRNA genes has been previously implicated in aberrant miRNA expression in several cancers , includiIncreased expression of both the −3p and −5p mature miRNAs was observed in Mino cells when compared to controls, and additionally, upregulation of −5p was observed in Raji and Toledo cells lines. Previous studies have identified miR-92a overexpression in BL, MCL, and DLBCL cell lines, and our study replicates these findings . Higher MIR17HG between cell lines, and between cell lines and healthy controls; however, this difference may not be biologically significant as the difference between mean methylation is within the detection limit margin of error (MIR17HG promoter region contribute to the differential miR-92a expression between NHL cell lines. A similar trend was identified in the NHL tumour samples; however, again the difference between mean methylation is within the detection limit margin of error. Aberrant methylation of the MIR17HG promoter has been previously correlated with dysfunctional expression of miRNAs in the miR-17–92 cluster in lung biopsies of individuals diagnosed with pulmonary fibrosis (MIR17HG CGI when compared to healthy controls. Although the mean difference between the two cohorts was not substantial, several individuals in the NHL patient cohort presented high levels of genomic DNA methylation across these targeted regions. Aberrant methylation of levels were observed in individuals diagnosed with specific subtypes of NHL when compared to controls, BL being the most prominent. An aggressive subtype of NHL, the BL cell line Raji was identified to exhibit increased methylation in these same regions in the cell line populations, and previous studies have reported a similar increase in methylation levels to those identified in this study in other miRNA promoter regions, in both BL cell lines and BL tumours (MIR17HG, and a previous study identified that aberrant methylation of miRNA promoter regions in circulating B-cells of CLL patients was associated with abnormal miRNA expression when compared to healthy B-cells. Increased expression of miR-92a has been implicated in CLL malignancy, specifically in lymph node proliferation centres (Differential methylation was observed in the upstream promoter CGI of of error . Therefofibrosis , and in fibrosis . The epi tumours . Increas centres . As the TET2 was seen across NHL cell lines and malignant tissues compared to controls, with specifically increased expression of TET2 in Mino, Raji, and Toledo cell lines alongside DLBCL and FL tumour tissue compared to controls. However, when taking into account the increased miR-92a-5p expression in the cell lines, NHL tumour tissue, and increased miR-92a-3p expression in the Mino cell line compared to controls, it is suggestive that miR-92a-5p and −3p may not be strong negative regulators of TET2 within these subtypes. Further assessment of the functional role of miR-92a on TET2 expression is required, feasibly with the utilisation of miRNA mimics and inhibitors, to determine the effect of miR-92a-5p and −3p expression and inhibition in NHL cell models.The findings of this study support previous conclusions regarding the significance of miR-92a in B-cell malignancy and reinforce the importance of investigating its specific role in NHL. Several previous studies have identified miR-92a as being implicated in dysregulation and suppression of tumour suppressors, including PTEN  and PHLPTET2 upstream promotor region when compared to controls and to Mino and Raji cell lines. This novel finding correlated with expected downregulation in TET2 expression in the SU-DHL-4 cells, along with a similar trend observed in the Raji cells compared to Mino and Toledo cell lines in previous studies (TET2 mRNA generally reported increased expression in cell lines and tumours, with the exception of SU-DHL-4, when compared to controls in our findings, TET2 methylation in our target region negatively correlated with expression in the NHL cell lines, indicating that the CpGs assayed in this region may be involved in the regulation of TET2 transcription and expression. TET2 overexpression in NHL is not well explored when compared to well-documented trends of downregulation in TET2 within various haematological malignancies (TET2 over-expression in CLL as observed in our study (TET2 overexpression in cells of some aggressive and indolent NHL subtypes is an innate immune response, involving tumour suppressor and DNA repair pathways, to the specific subtype of malignancy. Examination of TET2 protein and of downstream pathways of TET2 related expression regulation may assist in determining the translational consequence of TET2 overexpression in these NHL samples and cell lines.SU-DHL-4 and Raji cell lines showed significant hypermethylation in the studied CpGs within the  studies . Althouggnancies ; howeverur study . A possiTET2 variants in DLBCL as being associated with malignancy via the hypermethylation of pro-tumorigenic genes (TET2 gene in the specific NHL cell models and tumours samples may therefore provide greater understanding into the potential presence of disease-causing TET2 mutations, which may contribute to disease phenotypes; as highlighted by differential expression DLBCL cell lines SU-DHL-4 and Toledo, both of which are DLBCL cell models. DNA methylation studies of CpGs in the TET2 upstream promotor region across both NHL tumour-derived and control PBMC samples showed consistently low levels of methylation, and a similar trend was observed in the methylation of genomic DNA in the NHL case-control cohort. Differential methylation was observed in the TET2 regulatory region between the case and control cohort; however, this difference may again not be biologically significant (Previous studies have identified ic genes . Genotypnificant . It is tTET2 and miR-92a expression were considered (TET2 expression compared to other DLBCL and FL tumour samples, as well as compared controls considered (TET2 expression in this outlier is the presence of a functional mutation in TET2. Reed-Sternberg-like cells have been previously associated with TET2 mutations (TET2 is the cause of downregulated TET2 levels, thereby further supporting previous findings regarding TET2 as a potential marker and driver of malignancy in NHL.Clinical data from specific DLBCL tumour samples presented interesting characteristics and relationships when both nsidered . One tumnsidered . Hypermeutations  along wiutations . GenotypTET2 in NHL has been previously documented; however, the mechanisms driving TET2 expression regulation, such as DNA methylation, are not well explored. We identified in this study that aberrant methylation of several CpGs in the regulatory region of TET2 correlated with TET2 mRNA expression in NHL cell models. These findings are novel and may indicate that methylation of these regions may play a functional role in TET2 expression. This same relationship was not observed in NHL tumour samples or in NHL patient genomic DNA. Although a target of miR-92a, and aberrant miR-92a-3p and miR-92a-5p expression was observed in NHL cell models and NHL tumours, an association between miR-92a and TET2 was not observed in this study. Further assessment of the functional role of miR-92a-TET2 in human cell models is required; as is, ultimately, assessment in clinical cohorts. Although TET2 may not be comprehensively regulated by miR-92a, these novel findings regarding dysregulated methylation of TET2 regulatory regions in NHL is an intriguing relationship warranting further investigation and reinforces the prominence of TET2 as an element in NHL pathogenesis and malignancy.The role of