There are over 40 identified human disorders that involve certain proteins folding incorrectly, accumulating in the body causing damage to cells and organs and causing disease. Type 2 Diabetes Mellitus (T2DM) is one of these protein misfolding disorders (PMDs) and involves human islet amyloid polypeptide (hIAPP) misfolding and accumulating in parts of the body, primarily in the pancreas, causing damage to islet cells and affecting glucose regulation. In this review, we have summarised our current understanding of what causes hIAPP to misfold, what conformations are found in different parts of the body with a particular focus on what is known about the structure of hIAPP and how this links to T2DM. Understanding the molecular basis behind these misfolding events is essential for understanding the role of hIAPP to develop better therapeutics since type 2 diabetes currently affects over 4.9 million people in the United Kingdom alone and is predicted to increase as our population ages. Diabetes mellitus is the name given to a group of metabolic diseases characterised by chronic hyperglycaemia as a result of defective glucose regulation. Symptoms of diabetes and the severity can vary depending on the type of diabetes. Diabetes mellitus can be split into type 1 and type 2 diabetes. In type 1 diabetes mellitus  the pancreas is unable to produce any insulin. Type 2 diabetes mellitus (T2DM) usually occurs in adults and is due to the pancreas being unable to produce fully functioning insulin in sufficient quantities. Defective insulin action and/or secretion results in metabolic abnormalities and the associated chronic hyperglycaemia results in long-term damage leading to organ failure. During the early stages of T2DM, patients can be asymptomatic. Symptoms of chronic hyperglycaemia are polyuria, polydipsia, weight loss, polyphagia, and blurred vision in addition to growth impairment and susceptibility to infection . In addiDiabetes is a complex disease and is still not fully understood. Currently, there is no cure. Treatments simply alleviate the symptoms but do not stop the disease from progressing. Diabetes is a financial burden on health systems worldwide. Currently, in the United Kingdom alone, the cost for treatment, intervention, and complications are estimated to cost the National Health Service (NHS) £9.8 billion annually and is predicted to increase to £16.9 billion by 2035 . Hence, Proteinopathies or protein misfolding disorders, PMDs, are diseases resulting from incorrect protein folding, over 40 of which have now been identified . ProteinIn this review, we have reviewed the published structures of hIAPP in its different misfolded conformations  including mutagenic studies to try to understand how different parts of the protein and its different conformations are associated with disease. We have also summarised where these different conformations accumulate in the body and cause damage as it is now known that misfolded hIAPP accumulates in different parts of the body in addition to the pancreas. hIAPP has also been shown to interact with other amyloidogenic proteins accelerating their misfolding. This highlights the importance in understanding hIAPP not just to understand T2DM, but its role in related conditions such as AD and PD. Our review is novel since we have summarised all the mutations, all of the structures of hIAPP and locations of hIAPP deposits that have been published to date thus providing an up-to-date review that summarises what is known about hIAPP misfolding and aggregation and highlight gaps in our knowledge that urgently need addressing.2 and Cys7 is formed. It is then transported to the Golgi complex where it is packed into secretory granules and further cleaved to form a 37-amino acid peptide that is post-translationally modified by amidation at the C-terminal tyrosine. It is this active form that is co-secreted with insulin to regulate glucose levels in the body. IAPP is found in all mammals and healthy individuals and is co-produced with insulin in a molar ratio of 1:100 [HIAPP is expressed in the pancreatic β-cells of the Islets of Langerhans and belongs to the calcitonin family of peptides. IAPP acts in an endocrine as well as auto and paracrine manner involved in the regulation of glucose, bone resorption , renin aof 1:100 .6 and Thr9 yet the physiological role of glycans and their role in disease remains unclear.IAPP can undergo further post-translational modifications such as O-glycosylation  at Thr6 IAPP is normally soluble, monomeric, and natively unfolded in solution in its physiological form. It is described as an intrinsically disordered protein that has no regular three-dimensional structure  and is positively charged at physiological pH . IAPP is25, Ser28, Ser29, and do not form amyloid or T2DM. The S20G mutation which is highly amyloidogenic has been associated with early-onset T2DM and has been found in the Japanese and Chinese populations [Human IAPP is a highly amyloidogenic peptide and there is extensive evidence demonstrating that it can adopt different conformational states. Changes to the local environment, or in particular, changes in hydrophobicity to residues 20–29 , are thought to be crucial for amyloid formation . Sequenculations . IAPP isulations . In T2DMulations . This inThis ability of hIAPP to form amyloid like other PMDs and the observation that over 90% of T2DM patients have hIAPP deposits in the form of extracellular fibrillar aggregates in their pancreas  stronglyAmyloid can be defined as fibrillar deposits of proteins that contain a β-sheet structure and as a result, have enhanced dye-binding capacity to Thioflavin T , Congo RIn addition to being observed in the pancreas, misfolded forms of hIAPP have also been identified in different organs in T2DM patients , includiin vivo. The use of transgenic animal models has provided a longitudinal approach to support the correlation between IAPP aggregation and β-cell loss. Mice and rat IAPP do not aggregate, and these rodents do not normally develop diabetes. Animals where the human IAPP gene is highly expressed, develop diabetes as they grow during their lifetime [The likely event causing hyperglycaemia in T2DM is dysfunction or loss of β-cells leading to reduced secretion of insulin and development of insulin resistance . Here welifetime . The HIPlifetime . At 2 molifetime . Furtherlifetime .In vitro, hIAPP has a strong tendency to aggregate and will do so spontaneously within hours at µM concentrations [In vivo, IAPP is stored in secretory granules at mM concentrations [2+ and insulin at a slightly acidic pH prevents aggregation, especially during assembly of granule contents [in vitro and in vivo. Interaction with membranes can trigger a change in IAPP structure from the natural unfolded state to an α-helical structure that can insert into membranes [in vivo studies have identified oligomeric structures at the site of β-cell loss in pancreatic islets [trations . In vivotrations  but a cocontents ,56. The contents  and IAPPembranes ,58 or foembranes . Studiesembranes , and in c islets .2+ activation of calpain and apoptosis [There are several factors that could disturb proteostasis and induce toxic aggregation of IAPP inside β-cells. Intracellular toxic aggregates of IAPP were identified in biopsies of islets from patients with T2DM with a perinuclear or cytosolic distribution , the latpoptosis , disruptpoptosis  and disrpoptosis ,65.Overexpression of IAPP could activate the unfolded protein response (UPR). A series of three signalling pathways in the ER are triggered by IAPP protein misfolding and result in a local inflammatory response involving the release of pro-inflammatory cytokines IL-6, IL-8, MCP-1, and TNFα . MCP-1 hLarger amounts of oligomers have been found in β-cells of obese T2DM patients compared with non-obese patients, suggesting that other environmental factors such as oxidative stress can affect aggregation. Chronic hyperglycaemia or dyslipidaemia prevalent in T2DM will increase the stress responses . The β-c4 lower than in the pancreas. These findings were corroborated using HIP rat models where hIAPP is expressed only from the pancreas [A number of studies have explored the link between diabetes and neurological pathology mediated via IAPP. One of the first reports of hIAPP deposition in the brain reported that levels of oligomeric hIAPP were highest in brain homogenates taken from T2DM patients with dementia, compared with AD patients and controls . They alpancreas  demonstrpancreas . Loss ofpancreas .A study using brain samples from 2365 patients with and without diabetes, assessed AD neuropathology and concluded that diabetes is not associated with AD pathology . Diffusein vitro for Aβ 42 [There are, however, several studies that have shown a link between T2DM and AD. hIAPP deposits were found in brain blood vessels and associated parenchyma in patients with T2DM and AD some of which also included amyloid-β (Aβ). Interestingly, there were also hIAPP deposits in the AD group who had no apparent diabetes. The data suggested a model in which pancreatic hIAPP creates deposits in brain tissues where the initial aggregation events may be triggered by the high levels of hIAPP that occur in the prediabetic state. The results also demonstrate the potential for co-aggregation with other amyloidogenic proteins potentially influencing amyloid formation. These observations have been confirmed in other studies using animal models. Since rodent IAPP does not aggregate, this property is exploited in transgenic mice and rats expressing the aggregating human form of IAPP. A study using transgenic hIAPP mice focussed on the ability of α-synuclein to enhance amyloid formation involving co-aggregation with hIAPP in islets  using boor Aβ 42 , and α-sor Aβ 42 .Further work has focussed on the potential role of other factors that could modulate IAPP aggregation, in particular obesity or the pre-diabetic state. A positive correlation between brain amyloid deposits and levels of fasting plasma glucose, triglyceride, and HDL has been observed in cynomolgus monkeys  and in mDiabetes and hypertension, individually or combined are thought to account for 80% of all cases of end-stage renal disease. Nephropathy caused by diabetes is due to damage to blood vessels which leads to chronic kidney disease. A single study on humans has described deposits of IAPP in kidneys of patients with diabetic nephropathy (DN) . hIAPP d2) binding [hIAPP monomers have been detected in the plasma, white blood cells, and red blood cells in blood from a T2DM patient with nephropathy . In a co binding . hIAPP d binding . ElevateLarger conformers of misfolded hIAPP have also been identified in the blood of T2DM patients. Oligomers in the form of homodimers  and heterooligomers varying in size (1–400 nm) have been identified from sera of children with T2DM that were also present in obese children and those with T1DM . FibrilsDiabetes is known to be a risk factor for cardiovascular disease, attributed to the damage caused by high-glucose levels on vasculature supplying the heart. Here we consider whether IAPP could contribute to the risk of heart disease for diabetics by directly affecting the function of cardiomyocytes and surrounding cells. There are a very limited number of studies that have investigated IAPP aggregation in human heart tissue. Despa et al.  found IAIn a follow-up study  oligomerThese findings in patients suggest a correlation between IAPP aggregation, lipid oxidation, inflammatory stress, and myocyte damage, which was investigated using rat models and cultures of cardiomyocytes . Treatme2+ movements in HIP cardiomyocytes not found in UCD-T2M myocytes. Prediabetic HIP rats show signs of cardiac abnormality, with accumulation hIAPP, whereas UCD-T2M prediabetic rats do not.HIP rats were compared with the UCD-T2M rat model, which is not transgenic for hIAPP and in which diabetes develops due to β-cell dysfunction . In the 2+ movements in myocytes by hIAPP suggests an initial attachment to the sarcolemma, and then the formation of oligomers that could create cation-selective channels in the sarcolemma and also develop into fibrils. The membrane-attached fibrils could amplify structural alteration of membrane and hence Ca2+ dysregulation, leading to remodelling of myocytes via a Ca2+-dependent activation of HDAC NFAT transcription factors [A model of the disruption of Ca factors .Liu et al.  further 2+ signalling. The model was advantageous since other aspects of diabetes pathology such as hyperglycaemia or cardiac remodelling found in diabetes would be absent.Similar results have been found in mice perfused with aggregated hIAPP, which induced formation of hIAPP HNE– and MDA–adducts in the heart and activation of IL-1β. In a mouse knock-out model which does not express endogenous IAPP , adminisThese studies suggest that hIAPP can bind to cardiomyocytes, aggregate, and induce stress but it also seems possible that accumulation in vasculature of the heart could also contribute to compromising heart function.IAPP has been found in the eye of individuals with AD ,87. It iin vitro studies so the exact mechanism of misfolding in vivo remains unclear. IAPP amyloid formation is thought to occur via a nucleation-dependent polymerisation mechanism in vitro [Most of our information regarding the misfolding pathways come from in vitro . Evidencin vitro .The three main stages involved in amyloid formation are thought to include: (1) primary nucleation (lag phase) where small soluble oligomers are formed from monomeric peptides followed by (2) elongation (log phase) involving the formation of protofibrils, and fibril growth followed by (3) the plateau (or saturation) phase where mature amyloid fibrils dominate. hIAPP amyloid fibrils are further stabilised by their interactions with proteins such as serum amyloid P, apolipoprotein E, and perlecan . PerlecaGlycosaminoglycans such as heparan sulphate, are negatively charged polymers found in the extracellular matrix or on the membrane surface of cells. They have been found associated with hIAPP deposits in T2DM and are thought to be essential for amyloid formation . Lipid mLike many PMDs, there is still debate over what conformation of the amyloidogenic protein is associated with disease. Since fibrillar amyloid deposits of hIAPP have been observed in islet cells in the majority of T2DM patients , it is t18 and Tyr37 [In vitro experiments suggest that oligomers contribute to toxicity by disrupting cell membranes by forming pores that allow too many Ca2+ ions to enter that can lead to apoptosis [in vivo including studying their structure in more detail (if indeed they exist in vivo) to understand how they function in disease. Two-dimensional infrared spectroscopy experiments have revealed that an oligomeric intermediate form of hIAPP consists of parallel β-sheets [Oligomers ranging from dimers to hexamers have been identified during the early stages of aggregation  and in pnd Tyr37 . Residuend Tyr37 . Moleculnd Tyr37 . In vitrpoptosis . More evβ-sheets . Interesin vitro experiments suggest that fibrillisation of 1–37 is a synchronous process where protofibril elongation and mature fibril formation occur simultaneously [in vivo experiments are needed to corroborate these findings.Recent aneously  rather taneously . These sWhilst there is ample evidence linking hIAPP with T2DM, there is some evidence in the literature suggesting other proteins may misfold or somehow contribute to the misfolding of other proteins involved in T2DM. ProIAPP also appears to misfold since amyloid deposits composed of fibrillar aggregates containing proIAPP have been observed in secretory granules in some β-cells in human IAPP transgenic mice .Insulin degradation enzyme (IDE), which also degrades hIAPP , appearsFamilial mutations in ubiquitin carboxyl-terminal hydrolase L1 (UCH-L1) are associated with PD where the variant I93M is found to aggregate . UCH-L1 In vivo studies show that intravenous injection of Aβ fibrils into hIAPP transgenic mice cause increased amyloid formation in islet cells [Drosophila melanogaster models result in co-deposition of amyloid peptides [Aβ and α-synuclein, proteins that misfold in AD and PD respectively have been associated with IAPP misfolding and T2DM. et cells  and co-epeptides . A signipeptides .Understanding the structures of hIAPP assemblies is crucial for understanding exactly how IAPP is linked to T2DM. There are now many hIAPP structures that have been solved  many to Structures have revealed that monomers tend to have a predominantly α-helical content . The natbone fideex-vivo structures remain unknown.Fibrils tend to be characteristic of amyloid fibrils as seen in other PMDs usually being composed of two protofilaments and having a predominantly β-sheet content, (containing β-strands that run perpendicular to the fibre axis) that can be identified using X-ray fibre diffraction and cryo-electron microscopy (EM) . Like ot37 is consistent with the importance of amidation at the C-terminal thought to be important in fibril formation. The structures also explain one reason why the S20G hereditary mutation may facilitate hIAPP fibrillisation since glycine residues allow a more kinked conformation that in turn may allow the peptide chain to adopt a fibril-forming fold thus more easily forming a stable fibril structure. Since three of the polymorphs do not resemble any structures produced in vitro it suggests in vitro structures may differ from those in vivo as previously summarised for other amyloidogenic proteins [ex-vivo fibrils being more stable and protease-resistant which has important consequences for therapeutic design [A promising paper showed that hIAPP fibrils extracted from a T2DM patient could be used to seed synthetic hIAPP peptides to produce enough fibrils to study by cryo-EM . Four diproteins  due to ec design .Oligomers have been less well studied. Some research suggests they have a more α-helical content , whilst in vitro from peptides. Not all the structures are full-length, and the preparation methods used differ between studies. Hence, these factors may contribute to the different polymorphs produced as previously suggested [in vivo.Most of the structures solved to date are produced uggested  arguing We have summarised the current understanding behind the misfolding and aggregation of hIAPP and its link to T2DM in a simplified cartoon diagram shown in 5MGQ and 6Y1A), [Many mutational analysis experiments have been performed on hIAPP to understand the mechanism of hIAPP amyloid formation. To date, a large amount of data assessing the importance of certain residues in the sequence of hIAPP involved in amyloid formation have been collated. However, a systematic analysis of all possible mutations for positions of IAPP has not yet been published. Here we have summarised all mutational analysis studies that have been done to date using full-length (Supplementary File S1 and Table S1) or an 8–37 fragment of hIAPP (Supplementary File S1 and Table S2). Studies using shorter hIAPP fragments have been undertaken but are not included in this review as we deem them less physiologically relevant. Most of the studies summarised used hIAPP that is amidated at the C-terminal with point mutations altering a single amino acid at a time, to assess whether certain residues are involved in the ability of hIAPP to form amyloid (Supplementary Tables S1 and S2). For clarity, we have highlighted these residues in solved 3D structures of monomeric and fibrillar hIAPP (PDB codes: d 6Y1A),  as shownd 6Y1A), . These fd 6Y1A), .d-amino acid)) amyloid formation of hIAPP. Understanding the molecular basis behind the link between different residues and amyloidosis clearly requires further exploration [Mutating asparagine at position 14 can abolish  or accelerate (N14D) amyloid formation. Hydrophobic residues  abolish amyloid formation whereas mutating to a negatively charged aspartic acid accelerates amyloid formation ,145. Simloration .18. The protonation of histidine in an acidic environment results in the prevention of cation–π interactions and the formation of hydrogen bonds with other polar or charged residues. Whereas in a neutral environment, deprotonation of His18 favours these interactions [It should be noted that it is difficult to compare data between these different studies. This is due to different lengths of the protein being used (fragment vs full-length protein), the C-terminal amidation status differs and buffer type, buffer co-solutes, pH, ionic strength, and temperature used in the assays differ. In the truncated studies (Supplementary Table S2), even though the truncation is the first seven amino acids, outside the reported amyloid core, it may still affect the fibrillation rate and structures produced. Additionally, thioflavin-T binding assays used to assess hIAPP amyloid formation ,148 usedractions . In addiractions ,151. HenIn summary, from the studies summarised in Supplementary Tables S1 and S2, various mutations in the SRE conserved region  of IAPP 60IZ show similarities with hIAPP 7M61 (chain A), Aβ 2M4J with hIAPP 7M61 (chain B) and 50QV and 6SHS matching with hIAPP 7M62 highlighting residues 19–29 of hIAPP and 24–34 Aβ as having structurally similar regions that may be responsible for cross-seeding [In vivo, numerous studies have demonstrated that hIAPP can leave the pancreas and cross the blood–brain barrier and co-localise with Aβ in the brain contributing to the onset and progression of AD [There are numerous reports that hIAPP can interact with other amyloidogenic proteins promoting amyloidosis, thus linking T2DM to other disorders such as AD . This is-seeding . In vivoon of AD . The simon of AD .in vitro and in vivo in mice and human islet cells [in vitro could explain why having T2DM increases a person’s risk of getting PD but not the other way round since α-synuclein amyloid seeds actually inhibit IAPP amyloid production [T2DM has been linked to PD. Recently, α-synuclein and IAPP have been shown to co-aggregate et cells . The facoduction .Another amyloidogenic protein, the prion protein, PrP (fragment 106–126) and IAPP are reported to co-aggregate which is interesting since they lack sequence homology . Perhapsin vivo is needed for therapeutic design. Fibril formation is thought to involve a slow nucleation phase that may take many months or years as observed in other disorders such as AD [in vitro where hIAPP fibrils have been shown to occur in minutes [In addition to understanding the structure of hIAPP to high resolution, understanding the mechanism of amyloid formation ch as AD  where in minutes .Treatments for diabetes alleviate the symptoms but do not resolve the cause. Current treatments for diabetes include insulin, hIAPP analogues, biguanides, sulphonylureas, α-glucosidase inhibitors, dipeptidyl peptidase- 4 inhibitors, glucagon-like peptide receptor agonists, thiazolidinediones, SGLT2 inhibitors, and meglitinides .Destruction of β-cells is linked to hIAPP and insulin deficiency observed in T1DM and T2DM ,161, henAnother analogue called Davalintide, not yet approved, binds to the amylin receptor . HoweverKBP-088 is another DACRA and has been tested in clinical trials to treat weight loss and diabetes. Due to its high potency to bind both calcitonin and amylin receptors, it is a potent weight loss inducer. Using rats fed on a high-fat diet, those fed 5 μg/kg of KBP-088 per day for 11 weeks showed a 16% reduction in body weight . HoweverCagrilintide, which is a long-acting acylated IAPP analogue, is currently undergoing clinical trials for weight loss and glycaemic control in T2DM patients ,173. It Using hIAPP analogues in combination with other peptide hormones may accommodate the needs of individuals by mimicking the natural, energy homeostatic signalling pathways to create a specific desired physiological effect. For example, leptin, insulin, and peptide YY 3-36, a gut metabolic hormone, could be used in combination with an hIAPP analogue . Clearly25, Ser28, Ser29 in their IAPP sequence) form amyloidogenic forms of hIAPP and naturally get T2DM shows a direct link between the protein itself and T2DM.There are numerous studies that prove IAPP can form amyloid and a clear link between the accumulation of IAPP in T2DM patients and animal models strongly suggesting misfolded IAPP is linked to T2DM. The fact that only certain species (that have Ala2+ activation of calpain and apoptosis [There is extensive evidence linking dysfunction or loss of β-cells with reduced secretion of insulin and development of insulin resistance which is linked to IAPP amyloid formation . Howeverpoptosis , disruptpoptosis , disruptpoptosis ,65, and poptosis . The subpoptosis . Environpoptosis .ex vivo fibrils still remains unknown. In addition, the ex vivo/in vivo structure of smaller conformations of IAPP that precede fibrils is unknown. Understanding the structure of these conformers is key to developing therapeutics since fibrillar conformers are linked to late stages of T2DM so being able to prevent or slow fibril formation may prevent onset of the disease. A recent study showed that the molecular chaperone Hsp70 binds to oligomers but not monomers [As summarised in this review, evidence suggests that oligomers and fibrils are linked to T2DM  however monomers  hence exin vitro experiments. Using ex-vivo aggregates to seed fibrillisation reactions produced different conformations [It appears that hIAPP can form more than one conformer of fibrils and oligomers . This cormations  suggestirmations ,177 is uIAPP analogues such as Pramlintide, Davalintide, KBP-088, and Cagrilintide have all shown promise as a treatment for T2DM however issues with efficacy, solubility, and side effects need optimisation before they can be used as an effective T2DM treatment. Using natural compounds instead  or in coMore research is needed to identify what conformation hIAPP adopts when it deposits in different parts of the body  and in aT2DM is clearly a complex disease. Interestingly, not all T2DM patients have hIAPP deposits  and hIAPClick here for additional data file.


The curcumin analogues (3a–c) were tested for antiproliferative activity against a total of five dozen cancer cell lines in a single (10 µM) and five dose (0.001 to 100 µM) assays. 3,5-Bis(4-hydroxy-3-methoxystyryl)-1H-pyrazole-1-yl-(phenoxy)ethanone (3b) and 3,5-bis(4-hydroxy-3-methoxystyryl)-1H-pyrazole-1-yl-ethanone (3c) demonstrated the most promising antiproliferative activity against the cancer cell lines with growth inhibitions of 92.41% and 87.28%, respectively, in a high single dose of 10 µM and exhibited good antiproliferative activity (%GIs > 68%) against 54 out of 56 cancer cell lines and 54 out of 60 cell lines, respectively. The compound 3b and 3c demonstrated the most potent antiproliferative activity in a 5-dose assay with GI50 values ranging between 0.281 and 5.59 µM and 0.39 and 0.196 and 3.07 µM, respectively. The compound 3b demonstrated moderate selectivity against a leukemia panel with a selectivity ratio of 4.59. The HOMO-LUMO energy-gap (∆E) of the compounds in the order of 3a > 3b > 3c, was found to be in harmony with the anticancer activity in the order of 3c ≥ 3b > 3a. Following that, all of the curcumin analogues were molecular docked against EGFR, one of the most appealing targets for antiproliferative activity. In a molecular docking simulation, the ligand 3b exhibited three different types of interactions: H-bond, π-π-stacking and π-cationic. The ligand 3b displayed three H-bonds with the residues Met793 (with methoxy group), Lys875 (with phenolic group) and Asp855 (with methoxy group). The π-π-stacking interaction was observed between the phenyl (of phenoxy) and the residue Phe997, while π-cationic interaction was displayed between the phenyl (of curcumin) and the residue Arg841. Similarly, the ligand 3c displayed five H-bonds with the residue Met793 (with methoxy and phenolic groups), Lys845 (methoxy group), Cys797 (phenoxy oxygen), and Asp855 (phenolic group), as well as a halogen bond with residue Cys797 (chloro group). Furthermore, all the compound 3a–c demonstrated significant binding affinity  against the active site of EGFR. The curcumin analogues described in the current work might offer beneficial therapeutic intervention for the treatment and prevention of cancer. Future anticancer drug discovery programs can be expedited by further modifying these analogues to create new compounds with powerful anticancer potentials.With 19.3 million new cases and almost 10 million deaths in 2020, cancer has become a leading cause of death today. Curcumin and its analogues were found to have promising anticancer activity. Inspired by curcumin’s promising anticancer activity, we prepared three semi-synthetic analogues by chemically modifying the diketone function of curcumin to its pyrazole counterpart. The curcumin analogues ( Curcuma longa Linn.), belongs to the family Zingiberaceae is a β-diketone that showed an antiproliferative effect on various panels of cancer cell lines [2-), aryl side chain, diketone group, and carbon-carbon double bonds (-CH=CH-) to create a number of semi-synthetic curcumin analogues with improved bioactivity [The development of new antiproliferative agents with greater efficacy and a smaller amount of side effects remains a modern scientific research challenge. Over the last few decades, there has been a surge in interest in using natural products for therapeutic purposes. Although this concept appears to be new, our forefathers have used natural compounds/extracts to treat illness since time immemorial. The development of synthetic pharmaceuticals over the last century not only revolutionized modern medicine, but also accrued the undesirable properties and side effects associated with these drugs. The un-favorable properties of synthetic drugs have prompted a search for natural alternatives, with the hope that naturally occurring compounds will be better tolerated and safer than their synthesized counterparts ,2. Curcull lines . Medicinactivity . In thisactivity . Curcumiactivity ,7,8,9,10activity ,15,16,17activity ,19.Cancer is a disease in which a few of the body’s cells grow out of control and spread to other parts of the body. With 19.3 million new cases and almost 10 million deaths in 2020, cancer has become a leading cause of death today. Today, breast cancer is the most commonly diagnosed cancer, followed by lung cancer. Breast, lung, colorectal, liver, and stomach cancers account for 11.7, 11.4, 10, 7.3, and 5.6 percent of all new cancer cases, respectively . TobaccoThe EGFR is a key anticancer target and the most studied receptor in the tyrosine kinase super-family ,33. Curc3a–c) synthesized in the present investigation are summarized in 3a) was prepared by heating an equimolar mixture of curcumin (1)  and N-(2-methoxyphenyl)hydrazine carboxamide (2a)  in glacial acetic acid at 80 °C. Similarly, 1--4-hydroxy-3-methoxystyryl)-1H-pyrazol-1-yl)-2-(substituted phenoxy)ethan-1-ones (3b–c) were prepared by heating an equimolar mixture of curcumin (1)  and substituted phenoxy-acetohydrazide (2b–c) in glacial acetic acid at 80 °C. In this method (Method A), the reaction mixture was heated with continuous stirring for 10 h to complete the reaction while thin layer chromatography (TLC) in n-hexane: ethylacetate (6:4) was used to monitor the reaction throughout. The curcumin analogues were also prepared by another method (Method B). The method B glycerol-water system (1:2) was taken as solvent and the reaction was charged at 60–80 °C with continuous stirring for 6 h. The yields and time taken in the reaction are compared in 3a–c). The synthetic protocol is summarized in 2a, and 2b–c were prepared using the methods described elsewhere [3a–c) were confirmed by spectroscopic techniques. The IR spectra of curcumin analogues 3b–c showed acyl carbonyl (-CH2C=O) peak at 1650 and 1600 cm−1, while pyrazole C=N of compounds 3a–c was observed at 1544 to 1548 cm−1. The phenolic function (ArOH) of compounds 3a–c, while the phenoxy (-O-) functions of compounds 3b–c were observed ranging between 3427–3431 and 1276–1280 cm−1, respectively. The peak of the C-Cl function of 3c was observed at 680 cm−1. The 1H NMR showed a singlet for the six protons the methoxy function (OCH3) of curcumin in compounds 3a–c at δ 3.82–3.83 ppm, while the methoxy function (OCH3) present in the phenyl ring of compound 3a at δ 3.69 ppm. The methylene bridge (-CH2-) function of compounds 3b and 3c were observed at δ 4.60 and 4.80 ppm, respectively. The pyrazole CH of compounds 3a–c was observed as a singlet at 6.61 ppm and a broad singlet for the two protons of phenolic function (ArOH) at δ 9.14–9.91 ppm. The secondary amine peak of compounds 3a was observed as a singlet at δ 10.56 ppm. Two doublets were observed at δ 6.73–6.77 ppm corresponding to the -CH=CH- with coupling constant (J) of 12.4–14.4 Hz confirming the trans couplings. The aromatic protons of compounds 3a–c were observed as a singlet, and multiplet depending upon the nature of protons at δ 7.80–7.56 ppm. The 13C NMR of the prototype compound 3b displayed different carbon peaks at δ 160.71, 151.32, 147.46, 144.89, 137.31, 133.41, 129.83, 128.81, 123.6, 121.18, 120.19, 116.80, 114.31, 112.101, 107.72, 72.09, and 56.41 ppm. The ES-MS spectra of compound 3b showed a peak at m/z, 498.1 and 499.1 corresponding to M+ and (M + 1)+ corresponding to the molecular formula C29H26N2O6.The curcumin analogues  studies are critical for comprehending intermolecular dynamics and designing molecules with desired pharmaceutical characteristics. This tool may be used to identify a molecule’s fundamental properties such as its frontier molecular orbital energy levels, chemical reactivity, and stability, among others ,37. To f3a–c was determined to be 3.55, 3.40 and 3.35 eV, respectively. This is a typical value for tiny organic cores and corresponds to the prior study [As shown in the or study .3a displayed less antiproliferative activity and was found to displayed moderate activity against NCI-H522 and HT29 with growth percent (GP) of 73.19 and 80.40 percent, respectively. The compound 3b displayed maximum anticancer activity with growth percent (GP) 7.59 percent  followed by compound 3c  and 3a . The curcumin analogues 3b and 3c were found to be highly sensitive against all the leukemia cancer lines. The antiproliferative data of curcumin was retrieved from the NCI data base with NSC 32982 [3c showed a lethal effect on leukemia cell lines HL-60 (TB) (%GI = 118.70%) and RPMI-8226 (%GI = 106.80%), while the compound 3b displayed a lethal effect on RPMI-8226 (%GI = 103.54%). Similarly, the compounds 3b and 3c displayed a lethal effect on non-small lung cancer cell lines NCI-H522 with %GIs of 139.28 and 128.71, respectively. The compound 3b and 3c displayed more antiproliferative activity than curcumin against non-small lung cancer cell lines panel. The compounds 3b and 3c were found to be active against all colon cancer cell lines except COLO205 and HCC-2998 and a lethal effect was observed on the colon cancer cell line HT29 with %GIs of 133.23% by the compound 3b. The compounds 3b and 3c were also found to be active on all the CNS cancer cell lines, while lethal effect was observed against SF-539 (%GI = 110.32%) by compound 3b. The compounds 3b and 3c displayed more promising antiproliferative activity than curcumin against most of the colon and CNS cancer cell lines. The compound 3c exhibited a lethal effect on melanoma cell lines MDA-MB-435, SK-MEL-2, and SK-MEL-5 with %GIs of 112.15, 104.36 and 112.95 percent, respectively, while the compound 3b exhibited a lethal effect on melanoma cell line SK-MEL-2, and SK-MEL-5 with %GIs of 118.73, and 128.80, respectively. The compounds 3b and 3c were found to be active against all the melanoma cancer cell lines and also displayed superior antiproliferative activity to curcumin. The compound 3b exhibited a lethal effect on ovarian cancer cell lines OVCAR-3 and SK-OV-3 with %GIs of 104.46 and 128.58%, respectively. The compounds 3b and 3c also displayed superior antiproliferative activity to curcumin against ovarian cancer cell lines. The curcumin analogues 3b and 3c displayed promising antiproliferative activity against melanoma and ovarian cancer panels and their activity was found to be superior to curcumin. The compound 3b and 3c displayed a lethal effect on renal cancer cell lines with %GIs of 144.52 and 109.98 percent, while the compound 3b showed a lethal effect on breast cancer cell lines BT-549 and MDA-MB-468 with %GIs of 103.68 and 101.76 percent. Both the compounds were found to be active against all the cell lines of renal, prostate and breast cancer cell line panels with %GIs > 68 percent and displayed superior antiproliferative activity to curcumin. The mean GPs and %GIs showed that the curcumin analogues 3b and 3c showed superior antiproliferative activity to curcumin as shown in 3a–c) in term %GIs is given in the 3a–c) in term GP is given in the 3c demonstrated maximum sensitivity against NCI-H522 with GP of −28.71% (%GI = 128.71%) and least sensitivity against COLO205 with GP of 82.37% (%GI = 17.63%). The curcumin analogues 3b and 3c displayed promising antiproliferative activity in the one dose assay (10 µM). The compound 3b exhibited good inhibitions against 54 cancer cell lines out of 56 cancer cell lines, similarly the compound 3c exhibited good inhibitions against 54 cancer cell lines out of 60 cancer cell lines at 10 µM. The antiproliferative data of curcumin analogues (3a–c) in a single dose assay at 10 µM are given in 3b and 3c) demonstrated promising antiproliferative activity in a single dose assay, they qualified for 5-dose assay testing [The antiproliferative activity of the curcumin analogues was carried out against five dozen cancer cell lines derived from nine diverse panels including, breast, colon, CNS, leukemia, melanoma, non-small cell lung, ovarian, renal, and prostate cancer cell lines at one dose (10 µM) and five dose assay , as per the National Cancer Institute US ,40,41,42SC 32982 . The comSC 32982 ,44. The 3b demonstrated promising antiproliferative activity against 60 NCI cell lines with GI50 values ranging between 0.281 and 5.59 µM, TGI values ranging between 0.49 and >100 µM, and LC50 values 4.48 and >100 µM. Similarly, the compound 3c exhibited promising antiproliferative activity with GI50 values ranging between 0.224 and 3.82 µM, TGI values ranging between 0.837 and >100 µM, and LC50 values 11.4 and >100 µM. The antiproliferative activity of compounds 3b and 3c against 60 NCI cancer cell lines in the 5-dose screening in terms of GI50, TGI, and LC50 are given in 3b displayed the most promising antiproliferative activity against CCRF-CEM (GI50 = 0.281 µM), while the compound 3c displayed the most promising activity against SR (GI50 = 0.224 µM) among the leukemia cell lines. The compound 3b displayed the most promising antiproliferative activity against HOP-62 (GI50 = 1.08 µM), while the compound 3c displayed the most promising activity against NCI-H460 (GI50 = 0.517 µM) among the non-small lung cancer cell line. The compound 3b displayed the most promising antiproliferative activity against HCT-116 (GI50 = 0.386 µM), while the compound 3c displayed the most promising activity against HCT-15 (GI50 = 0.342 µM) among the colon cancer cell line. The compounds 3b and 3c displayed the most promising antiproliferative activity against the CNS cancer cell line SF-539 with GI50 values of 0.354 and 0.38 µM, respectively. The compounds 3b and 3c displayed the most promising antiproliferative activity against the melanoma cancer cell line MDA-MB-435 with GI50 values of 0.21 and 0.243 µM, respectively. The compounds 3b and 3c displayed the most promising antiproliferative activity against the ovarian cancer cell line OVCAR-3 with GI50 values of 0.552 and 0.511 µM, respectively. The compound 3b and 3c displayed the most promising antiproliferative activity against the renal cancer cell line UO-31 with GI50 values of 0.302 and 0.362 µM, respectively. The compounds 3b and 3c displayed the most promising antiproliferative activity against the prostate cancer cell line DU-145 with GI50 values of 1.37 and 0.833 µM, respectively. The compounds 3b and 3c displayed the most promising antiproliferative activity against the breast cancer cell line MCF7 with GI50 values of 0.343 and 0.336 µM, respectively. The compound 3b exhibited less selectivity against all the eight panels (except leukemia) of cancer cell lines with a selectivity ratio (SR) ranging between 0.59 and 1.63, similarly the compound 3c exhibited less selectivity against all the panels of cancer cell lines with SR ranging between 0.80 and 2.94  (a) and compared with that of the curcumin  implies a high chemical reactivity as well as biological activity [3c might possess a relatively high anticancer activity. The anticancer activity of 3b,c was found to be promising in a single dose assay with %GIs of 92.41 and 87.28, respectively, at 10 µM, while the mean GI50 was calculated as 1.241 and 1.149 µM, respectively, in the five dose assay. Both the compounds were found to be active against 54 cancer cell lines. Comparing the anticancer activity of the three compounds, the order followed as 3c ≥ 3b > 3a  and the lowest unoccupied molecular orbital (LUMO) ,48,49. Tactivity . This su 3b > 3a .3a displayed two H-bonds one between the phenolic function and the residue Leu718 and another between the carbonyl function and residue Thr854. The ligand 3c displayed four H-bonds and a halogen bond, the two H-bonds between one of the phenolic and methoxy functions and the most important residue Met793, while one H-bond was present between one of the phenolic functions and the residue Asp855 and one H-bond between the methoxy function and the residue Lys875. The 3D interaction of the ligands  is shown in 3b was found to be the most active compound in the series exhibiting three different types of interactions, including H-bond, π-π-stacking and π-cationic interactions. The ligand 3b displayed three H-bonds, one H-bond between the methoxy function and the most important residue Met793, one H-bond between another methoxy function and the residue Lys875 and one H-bond between the phenolic function and the residue Asp855. The π-π-stacking interaction was observed between phenyl (of phenoxy) and the residue Phe997, while π-cationic interaction was displayed between phenyl (of curcumin) and the residue Arg841. The 3D interactions of ligand 3b against the active site of EGFR are shown in The anti-EGFR activity of curcumin and curcumin analogues was well documented in the literature ,18. The 3a, which showed a mean percent GP of 99.22 percent [50 of 7.1 µM against MCF7 cell line [50 values of 5.6 ± 2.0, 5.9 ± 0.6 and 6.6 ± 1.9 µM, respectively [50 values ranging between 12.5 and 50 µM [50 values of 54.8 ± 2.5 and 52.1 ± 4.8 µM, respectively [50 values of 5.31, 8.33, 7.69, 8.62, and 8.19 µM and also inhibited Akt and STAT3 phosphorylation and increased ERK phosphorylation [50 value of 16 µM [3b and 3c) in the current investigation showed superior antiproliferative activity to curcumin in the one dose as well as the five dose assay. The curcumin analogues  displayed maximum sensitivity against the leukemia cell line panel with GI50 values ranging between 0.224 and 0.569 µM  displayed mean GPs of 7.59 and 12.72, respectively [50 ranging between 3.13 and 93.40 µM, while compounds 3b (GI50 = 0.281 µM) and 3c (GI50 = 1.34 µM) in the current investigation showed superior activity against the same cell lines [3b,c) were reported for the first time in the current investigation and they demonstrated promising antiproliferative activity against 5 dozen cancer cell lines derived from nine diverse panels. Some of the curcumin analogues reported earlier showed IC50 values of 16.71 (curcumin pyrazole) and 5.85 µM (curcumin semicarbazide) in an SRB assay against the HCT 116 cell line, while our compounds 3b and 3c displayed superior anticancer activity with GI50 values of 0.386 and 0.416 µM, respectively [3b was found to be moderately selective against the leukemia panel with an SR of 4.59. In our previous work the curcumin analogues were found to be non-selective towards the cancer cell line panels [3a–c) in the current investigation showed encouraging binding affinity against the EGFR, this information may also help to highlight the biological significance of curcumin analogues.The anticancer activity of six 3,5-bis(4-hydroxy-3-methylstyryl)-N-(substitutedphenyl)-1H-pyrazole-1-carboxamides has previously been reported, and their antiproliferative properties in terms of GPs ranged between 7.23 and −19.19 percent, which were found to be higher than that of compound  percent . The curell line . The curectively . The curnd 50 µM . The cucectively . The currylation . The curof 16 µM . The cur0.569 µM , while tectively ,55,59,60ll lines . The come panels ,64. Sinc1)  and N-(2-methoxyphenyl)-hydrazine carboxamide (2a)  in glacial acetic acid was stirred continuously at 80 °C in a sand bath for 10 h. The reaction mixture was then concentrated under a vacuum to remove excess solvent before being poured into crushed ice, filtered, dried, and recrystallized with ethanol to yield the compound 3a. The intermediate 2a was prepared as per the reported method in two steps starting from o-anisidine [An equimolar mixture of curcumin (nisidine . Method 1)  and substituted phenoxy acetohydrazide (2b–c) (1 mmol) in glacial acetic acid was stirred continuously at 80 °C in the sand bath for 10 h. The reaction mixture was then concentrated under a vacuum to remove excess solvent before being poured into crushed ice, filtered, dried, and recrystallized with ethanol to yield compound 3b–c. The intermediates 2b–c were prepared as per the reported method in two steps starting from substituted phenol as per the reported method [An equimolar mixture of curcumin (d method . Method http://www.chemaxon.com/ accessed on 24 July 2022), then transformed to a 3D structure and saved in xyz format. The DFT calculations were carried out in the gas phase using the Orca 5.03 package [https://www.chemcraftprog.com accessed on 24 July 2022) and Avogadro programs were used for analysis of the DFT results [The ligand’s 2D structure was created in Marvin Sketch  was evaluated against nine diverse panels of 60 cancer cell lines at 10 µM according to the National Cancer Institute (NCI US) protocol [3b–c) were treated against the cell lines in the given concentrations of 0.001 to 100 µM and there different parameters viz. GI50, TGI and LC50 were calculated for each cell line [50, TGI and LC50 are the molar concentration producing 50% growth inhibition, total growth inhibition (TGI) and a 50% cellular death, respectively.The antiproliferative activity of the curcumin analogues  [3a–c were saved as mol files, and docking was performed according to the reported protocol [The ligands 3a-c were molecular docked against EGFR. The protein data bank provided the EGFR (PDB: 3W2R) X-ray crystal structure with a resolution of 2.05 Å; R-value 0.220 (observed) (ly 2022) . The ligprotocol ,51.3a–c). The antiproliferative activity of compounds 3b and 3c showed promising anticancer activity in a one dose as well as a five dose assay. The compound 3b showed the most promising antiproliferative activity among the series of curcumin analogues and showed moderate selectivity against leukemia with an SR value of 4.59. The curcumin analogues  demonstrated superior antiproliferative activity to curcumin. HOMO-LUMO gaps (∆E) from DFT analysis and anticancer activities were discovered to be in agreement. All the curcumin analogues (3a–c) demonstrated significant binding affinity against the EGFR, a potential anticancer target. Because the compounds demonstrated promising anticancer activity and significant binding affinity against the EGFR, the current report has the potential to enhance the anticancer research development program in the future.We have prepared and reported the antiproliferative activity of three curcumin analogues (


ABCB1. We also identify functions for YB-1 in cell invasion, lipid metabolism and activation of the MYC oncoprotein. Importantly, through the generation of cell lines resistant to standard-of-care medulloblastoma therapies, we identify a drug-tolerant gene expression signature which may represent global, targetable mediators of medulloblastoma drug resistance. Together, our findings reveal important mechanisms and genes underlying therapy resistance in medulloblastoma and provide routes to their intervention.Medulloblastoma is the most common malignant childhood brain tumour. Under standard therapy, relapse occurs in 30% of patients and is almost universally fatal, accounting for 10% of all childhood cancer deaths. A barrier to effective medulloblastoma treatment is cellular resistance to standard-of-care therapies. Here, we investigate mechanisms surrounding medulloblastoma therapy resistance, both related to and independent from oncoprotein YB-1. Accordingly, we reveal roles for YB-1 in therapy sensitivity and the regulation of the multi-drug resistance gene ABCB1, as determined by chromatin immunoprecipitation. Whole transcriptome sequencing of YB-1 knockdown cells identified a role for YB-1 in the regulation of tumourigenic processes, including lipid metabolism, cell death and survival and MYC and mTOR pathways. Stable cisplatin- and vincristine-tolerant Group 3 and SHH cell lines were generated to identify additional mechanisms driving resistance to standard-of-care medulloblastoma therapy. Next-generation sequencing revealed a vastly different transcriptomic landscape following chronic drug exposure, including a drug-tolerant seven-gene expression signature, common to all sequenced drug-tolerant cell lines, representing therapeutically targetable genes implicated in the acquisition of drug tolerance. Our findings provide significant insight into mechanisms and genes underlying therapy resistance in medulloblastoma.Therapy resistance represents an unmet challenge in the treatment of medulloblastoma. Accordingly, the identification of targets that mark drug-resistant cell populations, or drive the proliferation of resistant cells, may improve treatment strategies. To address this, we undertook a targeted approach focused on the multi-functional transcription factor YB-1. Genetic knockdown of YB-1 in Group 3 medulloblastoma cell lines diminished cell invasion in 3D in vitro assays and increased sensitivity to standard-of-care chemotherapeutic vincristine and anti-cancer agents panobinostat and JQ1. For vincristine, this occurred in part by YB-1-mediated transcriptional regulation of multi-drug resistance gene  WNT), SHH , Group 3 (MBGroup3) and Group 4 (MBGroup4)—which can now be further categorised into second-generation sub-groups [Group3 is particularly poor due to the frequent occurrence of metastasis in this group, MBSHH and MBGroup4 have intermediate outcomes, whereas the outcome for MBWNT is generally good. Accordingly, improving our understanding of resistance mechanisms surrounding standard-of-care medulloblastoma chemotherapy represents an important area of medulloblastoma research [Medulloblastoma represents the most frequent malignant paediatric brain tumour. It comprises four principal molecular groups with different patterns of metastasis and overall prognosis—WNT  following therapeutic exposure, linking YB-1 expression to multidrug resistance mechanisms [Y-box binding protein 1 (YB-1), is a multi-functional protein encoded by the ively in ). Over-eively in , breast ively in , osteosaively in , head anively in , prostatively in , glioma ively in  and non-ively in . Frequenively in ). In tumively in ,13,14. Aively in ,16, whilively in ,18,19. Yively in ,21 and tively in , furtherchanisms .Group3 cell lines and subsequent transcriptomic analyses, the current study aimed to investigate the role of YB-1 in medulloblastoma tumourigenesis, with a focus on drug resistance. Bioinformatic analysis of publicly available medulloblastoma patient datasets was utilised to validate pre-clinical findings, whilst in vitro 3D invasion, cytotoxicity and chromatin immunoprecipitation assays provided key functional validation. Alongside this, we designed a global approach to identify alternative mechanisms driving resistance to standard-of-care therapies. Vincristine- and cisplatin-tolerant MBGroup3 and MBSHH cell lines were derived and mechanisms driving therapy resistance were identified by next-generation sequencing. Our findings, and the tools that we have created, identify key mediators of medulloblastoma drug resistance and routes to their intervention.The aforementioned studies highlight clear YB-1 involvement in cellular mechanisms surrounding stress response and drug resistance in multiple tumour types. However, how YB-1 is associated with such mechanisms in medulloblastoma remains to be explored. Through the creation of YB-1 knockdown MB2 and 37 °C. Mycoplasma testing was performed monthly using a PlasmoTestTM Mycoplasma Detection kit ; rep-pt1) as per the manufacturer’s instructions.DAOY and D283 cell lines were obtained from ATCC , ONS76 from Annette Künkele , UW-228-3 and D458 from John R. Silber , HD-MB03 from Till Milde  and CHLA-01 and CHLA-01R from Geoff Pilkington . DAOY, D283, D425, and D458 cells were cultured in DMEM with 10% foetal bovine serum , SH30541.03). UW-228-3 cells in DMEM/F-12 with 15% FBS and 1% sodium pyruvate. ONS76 and HD-MB03 cells were cultured in RPMI 1640 with 10% FBS. CHLA-01 and CHLA-01R were cultured in DMEM/F-12 supplemented with 2% B-27 , 17504044), 20 ng/mL recombinant human epidermal growth factor  and 20 ng/mL recombinant human basic fibroblast growth factor . All cell lines were grown under antibiotic-free culture conditions at 5% CO2 for 72 h. Following treatment, surviving cells were assayed with PrestoBlue , A13262) at a final dilution of 1:10 for 60 min at 37 °C and 5% CO2 and fluorescence was measured at 560/590 nm using a FLUOstar Omega ) microplate reader. The drug response was calculated as a percentage of the vehicle-treated control and dose–response curves and IC50 values were generated by non-linear regression analysis using GraphPad Prism 8 .For drug response analysis, cells were grown in standard culture conditions as previously described and harvested when they reached approximately 70% confluence. Cells were seeded at a density of 10,000 cells/well (D283), 5000 cells/well (HD-MB03 and D425) or 1000 cells/well (DAOY) in black-walled, clear-bottom 96-well plates and left to settle for 24 h. Cells were treated with cisplatin , S1166), vincristine , JQ1 , panobinostat  or an equivalent concentration of drug vehicle (dimethyl formamide (DMF) or dimethyl sulfoxide (DMSO))—and incubated at 37 °C and 5% CO2 and 37 °C and the treatment dose escalated upon cell proliferation. Cells were passaged in T-25 flasks in duplicate and dosing was commenced at 1/100 IC50 in each cell line. Matched DMF-treated (DMSO-treated for DAOY) vehicle control flasks were passaged bi-weekly alongside drug-treated flasks to account for morphological and genetic changes occurring from long-term culture and vehicle exposure. Drug resistance was measured by drug response assays, with cells considered resistant when the IC50 value of the treated cells had exceeded the treatment dose and the cell lines exhibited a significant increase in fold resistance .To generate drug-tolerant cell lines a continuous model of selection was utilised by which D283, D458 and HD-MB03 cell lines were cultured continually in the presence of cisplatin, and the DAOY cell line in the presence of vincristine, in antibiotic-free media at 5% CO® RNA Plus kit ; 740984). RNA was converted to cDNA using superscript II reverse transcriptase ; 1080-044). Gene expression of the resultant cDNA template was assessed by quantitative reverse transcription PCR ) using iQ SYBR SuperMix . Primer sequences were utilised as follows: YB-1 forward (5′ AAG AAG GTC ATC GCA ACG AAG 3′) and YB-1 reverse (5′ CTC CTA CAC TGC GAA GGT ACT 3′); ABCB1 forward (5′ CCC ATC ATT GCA ATA GCA GG 3′) and ABCB1 reverse (5′ GTT CAA ACT TCT GCT CCT GA 3′); ABCC1 forward (5′ TTC TCG GAA ACC ATC CAC GA 3′) and ABCC1 reverse (5′ CCT GTG ATC CAC CAG AAG GT 3′); GAPDH forward (5′ ATG TTC GTC ATG GGT GTG AA 3′) and GAPDH reverse (5′ CTC TTC TGG GTG GCA GTG AT 3′). The housekeeping gene GAPDH was used as a control to normalize the data and the relative mRNA expression level was calculated using ΔCq/ΔΔCq methodology [RNA isolation of cell lines was performed using the NucleoSpinhodology .g for 15 min at 4 °C and the resultant supernatant was harvested. The supernatant was mixed with 4 × SDS loading buffer and boiled at 95 °C for 10 min and subjected to SDS-PAGE on 12% SDS-PAGE gels. Following electro-blotting, samples were probed with the following antibodies: anti-YB-1 at 1:1000 ; 4202S); anti-GAPDH at 1:1000 ; HRP-linked anti-Rabbit IgG at 1:2000 . To visualise proteins, membranes were incubated with Pierce™ ECL Western Blotting Substrate ; 32106). Chemiluminescence was then measured using a LAS Mini 3000.Cells were lysed in NP-40 lysis buffer containing 1 × cOmplete™ EDTA-free Protease Inhibitor Cocktail ) on ice for 30 min with regular vortexing. Lysates were centrifuged at 14,000× ® mouse collagen IV  3410-010-02). Following 24 h, 50 µL of 100 µg/mL Laminin I  was added to each coated insert, which was partially dried for 1 h. 1 × 105 viable cells were then plated in serum-free media within the coated inserts and the outer chamber was filled with complete medium (10% FBS). Plates were incubated for 48 h at 37 °C and 5% CO2. Media was then removed from the chambers and invaded cells dislodged from the bottom surface of the insert by the addition of 1× cell dissociation solution for 1 h at 37 °C and 5% CO2 ; 3455-05-03). A PrestoBlue cell viability assay was used as previously described to assess metabolic activity and quantify cell migration relative to a standard curve of known cell numbers.Prior to assay commencement, cells were starved in reduced-serum (2% FBS) media, and 8 µm 24-well plate transwell inserts ; 662641) were coated with 50 µL 10 µg/mL CultrexYBX1 ; VGH5526-EG4904) according to the manufacturer’s instructions.Cell lines with stable knockdown of YB-1 expression were generated through shRNA-mediated gene silencing using the GIPZ Lentiviral particle starter kit for ABCB1 promoter region was undertaken. Primer sequences were as follows: ABCB1 forward (5′ CAT GCT GAA GAA AGA CCA CTG C 3′) and ABCB1 reverse (5′ AGG CTT CCT GTG GCA AAG AG 3′).D283 and HD-MB03 cells were cross-linked in 1% paraformaldehyde for 10 min. Cell lysis, shearing, immunoprecipitation and DNA purification were undertaken using a Magna ChIP A/G immunoprecipitation kit ; 17-10085) according to the manufacturer’s instructions. Samples were sonicated using a water bath sonicator  to obtain 200–500 bp chromatin fragments. The following antibodies were utilised to pull down protein-DNA complexes, with Immunoglobin (IgG) 1 used as a negative control and histone H3K4Me3 used to confirm active promoter regions: YB-1 ; SC101198); IgG1 ; H3K4me3 ; 61379). To quantify target protein interaction with the gene of interest, qPCR using primers specific to an inverted CCAAT box within the 2 fold change ≥0.5 or ≤−0.5, p-value ≤ 0.05 and False Discovery Rate (FDR) p-value ≤ 0.1 (p < 0.05 with the Benjamini-Hochberg (B-H) method employed to control FDR. Where possible, Z-score activation predictions were calculated. Z-scores > 2 or <−2 were considered as significantly activated or inhibited, respectively.YB-1 knockdown and non-silencing control lines were pelleted in triplicate in consecutive passages and RNA was extracted using the RNeasy Mini kit  according to the manufacturer’s instructions. Whole transcriptome sequencing was performed by Qiagen Genomic Services . Library preparation was undertaken using the QIAseq Stranded Total RNA Library Kit  with QIAseq FastSelect rRNA/globin depletion according to the manufacturer’s instructions. Libraries were sequenced on a NextSeq 500 instrument ; SY-415-1002) in a dual index 1 × 75 bp format at a sequencing depth of 30 M reads/sample. Sequencing data analysis was conducted using CLC Genomics Workbench  and CLC Genomics Server . Differentially expressed genes (DEGs) were defined as logue ≤ 0.1 . Ingenui2 fold change ≥0.5 or ≤−0.5 and B-H-adjusted p-value ≤ 0.05. Gene ontology enrichment analysis of differentially expressed genes was conducted with R package GOSeq (version 1.4.0) with default settings and a cut-off p-value of p ≤ 0.05.Drug-tolerant cell lines were pelleted in triplicate in consecutive passages and RNA was extracted as previously described. Libraries were prepared using the QuantSeq 3′mRNA-Seq library prep kit for Illumina in the forward read direction supplemented with unique molecular index (UMI) as per manufacturer’s instructions ; 015.96). The resultant library pool was sequenced on a NextSeq 500 High Output v2.5 75 cycle kit ; 20024906), to generate approximately 5 million 75 bp single-end reads per sample. Differential gene expression analysis was undertaken using the R statistical environment package DESeq2 (version 1.24.0) with default settings. DEGs were defined as LogResults are shown as mean ± SEM of the indicated number of independent experiments. The statistical significance of differences in group results was compared using one- or two-way analysis of variance (ANOVA) with multiple comparison testing as indicated. All statistical analyses and plots were carried out using GraphPad Prism 8  unless stated otherwise. The number of biological samples, corresponding statistical test and significance levels are indicated in each figure legend.http://r2.amc.nl (accessed on 6 April 2020)). A cut-off between high YB-1 gene expression and low YB-1 gene expression groups was selected, where p-values obtained from the log-rank test were minimized.Published medulloblastoma patient datasets as described by Cavalli et al. , NorthcoGroup3, MBGroup4 and MBSHH patients with high expression levels of YB-1 mRNA had significantly worse 10-year overall survival outcomes than those with low YB-1 expression levels -like barrier. Cells were seeded in serum-free media in the upper chamber of the assay and incubated for 48 h to facilitate migration (uncoated membrane), or invasion (coated membrane), towards a complete media-containing lower chamber C. AbundaComparatively, no alteration in migration was observed upon YB-1 knockdown in either cell line D, indicaGroup3 status of each line. Notable top 10 DEGs included those encoding extracellular matrix components COL8A1 and TNC and cell adhesion molecule CADM3, which have previously been associated with cellular invasion and migration in gastric cancer [We next wanted to build a picture of the global YB-1 transcriptome in Group 3 medulloblastoma. Accordingly, we performed whole transcriptome sequencing on each YB-1 knockdown line and the appropriate non-silencing control line. Analysis of the most significantly altered genes (up and down) confirmed YB-1 to be down-regulated in both cell lines  was employed to predict affected cellular biology from statistically significant differential gene expression patterns. Despite no commonality at a gene level, analysis of affected molecular and cellular functions identified cell death and survival processes to be dysregulated in both cell lines following YB-1 knockdown C,D. ExamIPA molecular and cellular functions analysis also revealed enrichment for metabolic processes, with alterations in carbohydrate metabolism specific to HD-MB03, vitamin and mineral metabolism specific to D283 and lipid metabolism affected across both cell lines, C,D. ExamMYC/MYCN amplification with treatment-refractory disease and relapse in medulloblastoma [MYC-amplified status of this line, which may act to mask such changes in MYC activation. Interestingly, upstream regulator analysis also revealed inhibition of rapamycin-insensitive companion of mTOR (RICTOR) and regulatory-associated protein of mTOR (RPTOR)—components of the mechanistic target of rapamycin (mTOR) signalling. Previous studies have demonstrated clear communication between YB-1 and the PI3K/AKT/mTOR pathway and thus this finding may indicate a similar association within paediatric brain tumours [Group3, revealing potential novel functions in cellular stress response, lipid metabolism, as well as MYC and mTOR pathway activity.To further analyse the YB-1 transcriptome in Group 3 medulloblastoma, we also performed upstream regulator analysis, a subset of IPA that allows the identification of upstream regulators that may be responsible for the gene expression changes in a particular dataset F. The beblastoma ,35. The  tumours ,37,38. TYB-1 appears to be implicated in cell death and survival processes and cellular stress mechanisms A–F. ThisGroup3 [ABCB1 exon 3 promoter region within both cell lines . Contrastingly, MBSHH cell line DAOY did develop vincristine tolerance (DT-DAOY-VIN), displaying a significant 2.8-fold increase in IC50 compared to the parental line  were generated utilising standard-of-care medulloblastoma chemotherapy agents: cisplatin and vincristine. Following continuous drug treatment and dose escalation following cell proliferation, three cisplatin-tolerant MBtal line D. It is is study . Due to ABCB1 compared to its vehicle-treated control sister line , despite cisplatin being a non-ABCC1 substrate; perhaps indicative of a function of ABCC1 in therapy resistance and/or cell survival independent of its role in cytotoxic drug efflux [Assessment of ABC transporter expression supported this theory, with the DT-DAOY-VIN line exhibiting significantly elevated expression of DAOY-VIN . InteresThe aforementioned drug-tolerant cell lines underwent 3′mRNA sequencing to elucidate transcriptomic alterations that occur following chronic drug exposure and the concurrent development of drug tolerance. Unsupervised hierarchical clustering analysis revealed significant alteration in the transcriptome following the acquisition of drug tolerance . InteresGroup3 lines , only one, LGALS1, had been researched previously in relation to medulloblastoma, where it is up-regulated in MBSHH patients and activated by the SHH signalling pathway [YB-1 independent mechanisms of drug resistance were revealed by 3′mRNA sequencing of three cisplatin-tolerant MB pathway . Thus, tABCB1 and provide evidence to support a function for YB-1 in intrinsic cellular resistance to various anti-cancer therapies, both related to and separate from its aforementioned role as a regulator of the ABCB1 gene. Finally, through the establishment and next-generation sequencing of drug-tolerant medulloblastoma cell lines, we identify targetable hits implicated in the acquisition of tolerance to standard-of-care agents, which may inform future pre-clinical investigations with a focus on hindering the development of therapy resistance.In conclusion, we demonstrate here an association between YB-1 expression and various aspects of medulloblastoma tumourigenesis, including cell invasion, MYC oncoprotein activity and lipid metabolism. Bioinformatic analysis of published datasets validated these pre-clinical findings, supporting a role for YB-1 in cell death and survival processes, cholesterol biosynthesis, response to therapeutics and MYC/mTOR signalling; as well as overall survival and metastasis in medulloblastoma patients. Importantly, we also identify YB-1 as a transcriptional regulator of drug resistance-related gene


Atypical teratoid rhabdoid tumor (ATRT) is a deadly type of human pediatric brain cancer without effective treatments. ATRT is mainly linked to the inactivation of a tumor suppressor gene, SMARCB1; however, additional biomarkers remain to be identified to develop novel therapeutic strategies. Therefore, different tumor antigens and extracellular matrix modulators were investigated in two human ATRT and one kidney malignant rhabdoid tumor cell lines and compared with the nonmalignant HEK293 cell line. Alpha-fetoprotein (AFP), mucin-16 (MUC16 or cancer antigen 125/CA125), osteopontin (OPN), and mesothelin (MSLN) are highly expressed in these human malignant rhabdoid cancer cell lines. Inhibiting MMPs using a small-molecule inhibitor decreased cell survival. This biomarker discovery process may lead to the identification of novel diagnostic and therapeutic strategies, such as the development of targeted and immunotherapies against cancer biomarkers, to treat cancer patients.Introduction: Atypical teratoid rhabdoid tumor (ATRT) is a lethal type of malignant rhabdoid tumor in the brain, seen mostly in children under two years old. ATRT is mainly linked to the biallelic inactivation of the SMARCB1 gene. To understand the deadly characteristics of ATRT and develop novel diagnostic and immunotherapy strategies for the treatment of ATRT, this study investigated tumor antigens, such as alpha-fetoprotein (AFP), mucin-16 (MUC16/CA125), and osteopontin (OPN), and extracellular matrix modulators, such as matrix metalloproteinases (MMPs), in different human malignant rhabdoid tumor cell lines. In addition, the roles of MMPs were also examined. Materials and methods: Five human cell lines were chosen for this study, including two ATRT cell lines, CHLA-02-ATRT and CHLA-05-ATRT; a kidney malignant rhabdoid tumor cell line, G401; and two control cell lines, human embryonic kidney HEK293 and HEK293T. Both ATRT cell lines were treated with a broad-spectrum MMP inhibitor, GM6001, to investigate the effect of MMPs on cell proliferation, viability, and expression of tumor antigens and biomarkers. Gene expression was examined using a reverse transcription polymerase chain reaction (RT-PCR), and protein expression was characterized by immunocytochemistry and flow cytometry. Results: All the rhabdoid tumor cell lines tested had high gene expression levels of MUC16, OPN, AFP, and MSLN. Low expression levels of neuron-specific enolase (ENO2) by the two ATRT cell lines demonstrated their lack of neuronal genotype. Membrane-type 1 matrix metalloproteinase (MT1-MMP/MMP-14) and tissue inhibitor of metalloproteinases-2 (TIMP-2) were highly expressed in these malignant rhabdoid tumor cells, indicating their invasive phenotypes. GM6001 significantly decreased ATRT cell proliferation and the gene expression of MSLN, OPN, and several mesenchymal markers, suggesting that inhibition of MMPs may reduce the aggressiveness of rhabdoid cancer cells. Conclusion: The results obtained from this study may advance our knowledge of the molecular landscapes of human malignant rhabdoid tumors and their biomarkers for effective diagnosis and treatment. This work analyzed the expression of human malignant rhabdoid tumor antigens that may serve as biomarkers for the development of novel therapeutic strategies, such as cancer vaccines and targeted and immunotherapies targeting osteopontin and mesothelin, for the treatment of patients with ATRT and other malignant rhabdoid tumors. Malignant rhabdoid tumors are aggressive tumors that mostly appear in children under two years of age . AtypicaATRT has several histological features, including eosinophilic cytoplasm, large nucleoli, and filamentous cytoplasmic inclusions ; howeverCD44 is expressed on the surface of ATRT cells and tumor mesenchymal stromal cells from the rhabdoid tumor microenvironment . In addiCompared with medulloblastoma, ATRT has a stemness-like feature and expresses an embryonic stem cell gene profile of OCT4, NANOG, SOX2, and c-MYC . Hence, Matrix metalloproteinases (MMPs) are zinc-dependent endopeptidases/proteinases with the function of degrading extracellular matrix (ECM) proteins, including basement membrane and interstitial matrix . Their rThe expression levels of tissue inhibitors of metalloproteinases (TIMPs) and disintegrin and metalloproteinases (ADAMs) were also measured in the five cell lines. ADAMs are anchored on the cell membrane, functioning in processes of fertilization, restenosis, neurogenesis, and tumor invasion and metastasis by degrading ECM ,58, wherDiagnostic and therapeutic biomarkers with improved sensitivity and specificity are needed for ATRT due to its complexity ,61. ATRT® CRL-3020TM, ATCC) and CHLA-05-ATRT  cells were cultured based on the manufacturer’s protocol. Briefly, the cells were plated in suspension using Gibco Dulbecco’s Modified Eagle Medium: Nutrient Mixture F-12  supplemented with 2% B-27, 20 ng/mL of epidermal growth factor , and 20 ng/mL of fibroblast growth factor (FGF)-2 , in a non-treated 6-well plate at 37 °C, 5% CO2. The initial cell density was 1 × 106 cells/mL. The cell density was determined every 2 to 3 days using a hemocytometer (see 6 cells/mL), they were passaged at the ratio of 1:6 into a new vessel.Both CHLA-02-ATRT  and HEK293T  cells were cultured in DMEM supplemented with 10% fetal bovine serum (FBS) at 37 °C at 5% CO2. The cells were passaged when they reached 80% confluence using trypsin . G401  was cultured based on the ATCC protocol. Briefly, the cells were cultured in McCoy’s 5A Medium  supplemented with 10% FBS. The cells were also passaged upon reaching 80% confluence.HEK293  was used to quench the reaction. A mixture of the cell solution with Trypan Blue 0.4%  at a 1:1 ratio was pipetted onto Countess chamber slides  using the Countess® II FL instrument . The total cell number and cell viability were reported.Both CHLA-02 and CHLA-05 were harvested from suspension and washed three times with PBS. Then, the samples were treated with Accumax  for 20 min at 37 °C at 5% COMatrigelTM-coated surfaces to let the cells spread outward from the spheroids. The images of these cells were also taken using an Olympus IX70 microscope.The images of CHLA-02 and CHLA-05 aggregates were taken using an Olympus IX70 microscope . In addition, some of the aggregates of CHLA-02 and CHLA-05 were replated on ® Total RNA Kit I  was used to isolate the total RNA from each cell line, and an RNA Clean & Concentrator-5 kit  was used to clean and concentrate the total mRNA samples. The reverse transcription required 2 μg of the total RNA, anchored oligo-dT primers , and Superscript III  and was performed according to the manufacturer’s protocol. The primers specific to target genes (ACTB) gene was used as an endogenous control for normalizing expression levels. RT-PCR was performed on an ABI7500 instrument  using SYBR1 Green PCR Master Mix (Applied Biosystems). The amplification reactions were 2 min at 50 °C, 10 min at 95 °C, and 40 cycles of 95 °C for 15 s, 55 °C for 30 s, and 68 °C for 30 s. The fold variation in gene expression was quantified using the comparative 2−ΔΔCT method based on the comparison of expression of the target genes  among different conditions.An E.Z.N.A.et genes  were desMatrigelTM-coated 96-well plate and then fixed with 4% paraformaldehyde (PFA). For nuclear markers, the cells were permeabilized with 0.5% Triton X-100. The samples were then blocked in 2% FBS in phosphate-buffered saline (PBS) for 1 h and incubated with various mouse antibodies in a blocking buffer at 4 µg/mL . The data were analyzed against the isotype control using FlowJo software.Briefly, 1 × 106 cells/mL. GM6001  was dissolved in DMSO to a stock concentration of 8 mM and added to the culture at a final concentration of 10 µM [Matrigel. In addition, the cells were harvested for RT-PCR analysis for epithelial, mesenchymal, and important ATRT biomarkers .CHLA-02 and CHLA-05 cells were replated at a density of 1.6 × 10of 10 µM . After 4n = 3). To compare two conditions in t-test was used to determine the significance. A p-value < 0.05 was considered significant. On the other hand, for comparing more than two different conditions   A–D. The for ATRT , and theImmunocytochemistry experiments were performed to detect the protein expression of several markers . AFP wasMMPs), tissue inhibitors of metalloproteinases (TIMPs), and a disintegrin and metalloproteinases (ADAMs) were characterized for all five cell lines  and mesenchymal markers . The epithelial gene expression of CDH1 was higher in all the cell lines when compared with HEK293 cells, and it was highest in G401 cells   A,B. In aeatment) C. Despiteatment) D.CDH1) and most mesenchymal markers . The important ARTR biomarkers (MSLN and OPN) were also decreased by 16%. CHLA-05 showed an increase in the expression of VIM (~4 folds) and MUC16 (~0.14 folds).The effect of GM6001 on different biomarkers in both ATRT cell lines was also investigated using the RT-PCR method . The preThe two human ATRT cell lines, CHLA-02 and CHLA-05, showed distinct differences in several gene expression markers. In many cases, the investigated MMP, TIMP, and ADAM gene expression levels in CHLA-02 were higher than in CHLA-05 , suggestCD44 in CHLA-02 might suggest its invasive phenotype. AFP gene and protein expression may serve as a valuable biomarker for malignant rhabdoid tumors due to its much higher expression levels compared to nonmalignant cells.Among all the biomarkers investigated, OPN, AFP, MUC16, and MSLN may be the most important biomarkers for ATRT. OPN is a stem cell-promoting factor in glioblastoma , and itsMSLN mRNA, but only low levels of protein were detected by immunocytochemistry (ICC), indicating that MSLN protein expression might be low, the proteins might be shed into the cell culture media, or that the sensitivity of the antibody is not sufficient. Therefore, targeting the ERK and AKT pathways and disrupting MSLN–MUC16 interactions may become novel strategies for ATRT treatment.MUC16 acts as a signaling molecule and a lubrication barrier of the epithelial surface . The expNMP1 gene expression was lower in both ARTR cell lines compared with the nonmalignant HEK293 cells in this study. This could be due to the different isoforms of NMP1. OCT4 is expressed in other cancer types, such as glioma, hepatocellular carcinoma, rectal cancer, and pancreatic cancer [OCT4 mRNA in rhabdoid tumor cell lines, but the results were inconsistent with ATRT cell lines. Our results showed that gene expression levels were significantly higher in G401 than in HEK293 cells. At the protein level, all the rhabdoid tumor cell lines expressed OCT4, suggesting its important role in preserving the stem cell-like property. However, in other studies, the protein expression of OCT4 was not detected in ATRT or non-central nervous system malignant rhabdoid tumors [Our NPM1 and OCT4 expression results differed from those reported in the literature. Even though NMP1 has been suggested to be a potential target for ATRT treatment in the literature , NMP1 gec cancer ,77,78,79d tumors ,81,82,83MMP-26, an invasion biomarker in various cancers, including gliomas [MMP7 is a biomarker associated with the epithelial-to-mesenchymal transition (EMT) of tumor cells that are highly invasive [MMP7 was highly expressed in CHLA-02. The gene expression profile of CHLA-02 demonstrated this cell line’s aggressive and invasive phenotype.Investigating MMP and TIMP expression is important for understanding the invasiveness of ATRT cells. MMP1 is highly expressed in glioblastoma multiforme xenografts . Further gliomas , astrocy gliomas , colorec gliomas , and eso gliomas . High MM gliomas . MMP26 m gliomas ,90 and i gliomas ,92 to prinvasive ,94, as winvasive  and glioinvasive . MMP7 waTIMP1 or 2 [TIMP2 in both ATRT cell lines was significantly higher than in the nonmalignant HEK293 cell line. TIMP4, a biomarker for astrocytoma in glioma patients [LOX) and SNAI2 were highly expressed in both ATRT cell lines. LOX and SNAI2 can regulate the induction of TIMP-4, which can lead to tumor invasion, migration, and VEGF expression [In addition to their inhibitory abilities against MMPs, TIMPs may promote cell proliferation and inhibit apoptosis ; howeverMP1 or 2 . This stpatients , was higpatients . Lysyl Opression .ADAM9 was highly expressed in both ATRT cell lines, but ADAM10 was only expressed in CHLA-02. ADAM9 is an adhesive protein and can induce cell signaling, leading to increased proteolytic activities [tivities  and it itivities ,104. ADAtivities ,106,107,tivities ,108. ADAtivities ,110. It tivities ,112. Mortivities ,114.Matrigel-coated surfaces, further supporting this hybrid phenotype hypothesis. Recently, two chromatin-modifying complexes, polycomb repressive complex 2 (PRC2) and lysine methyltransferase 2D-complex of proteins associated with SET1 (KMT2D-COMPASS), have been suggested to govern epithelial–mesenchymal plasticity [Epithelial–mesenchymal transition (EMT) is a process in which cells change their phenotypes from polarized epithelial to mesenchymal ,116. Thiasticity . Thus, Aasticity . Therefoasticity .VIM expression in CHLA-05. The suppression of MYC protein expression in colorectal cells by knocking down MMP1 [GM6001 is a potent broad-spectrum MMP inhibitor. GM6001 treatment decreased the cell proliferation and gene expression of several important biomarkers in both CHLA-02 and CHLA-05, including MSLN, OPN, and many mesenchymal markers. The effect of GM6001 was more significant in CHLA-02, perhaps due to the higher MYC and MMP expression levels in CHLA-02 than in CHLA-05 . In addiown MMP1  may suggHuman embryonic kidney cell line HEK293 cells were used as nonmalignant controls for G401, a kidney-derived malignant rhabdoid tumor; however, HEK293 may not be the ideal control for ATRT cell lines. The origin of ATRT is still under investigation, but it might be in neural progenitor cells ; howeverThis comprehensive study investigated biomarkers of different types, epithelial, mesenchymal, MMPs, TIMPs, and ADAMs, as well as the effects of GM6001 on ATRT cells; however, these studies were limited to using two ATRT cell lines, one malignant kidney rhabdoid tumor cell line (G401), and two kidney control cell lines HEK293 and HEK293T. In the future, other ATRT cell lines and patient-derived xenograft samples need to be investigated to verify these ATRT antigens. Although CHLA-02 and CHLA-05 have different cellular pathways and metabolic regulations, they might not be able to represent all ATRT patient-derived cell lines and tissues. Thus, future large-scale studies are needed to verify our findings for therapeutic development to treat patients with ATRT and other malignant rhabdoid tumors.In summary, CHLA-02 and CHLA-05 may represent two different subtypes of ATRT based on their gene expression profiles. AFP, MSLN, MUC16, OPN, and MT1-MMP/MMP14 may serve as biomarkers for human malignant rhabdoid tumor detection and therapeutic targets. Due to their high MT1-MMP and other MMP expression levels, ATRT cells may be highly invasive. Treating ATRT with a broad-spectrum MMP inhibitor, GM6001, decreased cell proliferation with no significant changes in viability or several important markers in both ATRT cell lines. ATRT may utilize the MYC and ERK pathways as their central metabolic regulation, suggesting an important therapeutic intervention approach. OPN and MSLN may serve as targets for immunotherapies and cancer vaccine development to treat ATRT patients. This study may also advance our knowledge of the molecular features of malignant rhabdoid tumors and their aggressiveness.


Hepatic stellate cells (HSCs) are also known as lipocytes, fat-storing cells, perisinusoidal cells, or Ito cells. These liver-specific mesenchymal cells represent about 5% to 8% of all liver cells, playing a key role in maintaining the microenvironment of the hepatic sinusoid. Upon chronic liver injury or in primary culture, these cells become activated and transdifferentiate into a contractile phenotype, i.e., the myofibroblast, capable of producing and secreting large quantities of extracellular matrix compounds. Based on their central role in the initiation and progression of chronic liver diseases, cultured HSCs are valuable in vitro tools to study molecular and cellular aspects of liver diseases. However, the isolation of these cells requires special equipment, trained personnel, and in some cases needs approval from respective authorities. To overcome these limitations, several immortalized HSC lines were established. One of these cell lines is CFSC, which was originally established from cirrhotic rat livers induced by carbon tetrachloride. First introduced in 1991, this cell line and derivatives thereof  are now used in many laboratories as an established in vitro HSC model. We here describe molecular features that are suitable for cell authentication. Importantly, chromosome banding and multicolor spectral karyotyping (SKY) analysis demonstrate that the CFSC-2G genome has accumulated extensive chromosome rearrangements and most chromosomes exist in multiple copies producing a pseudo-triploid karyotype. Furthermore, our study documents a defined short tandem repeat (STR) profile including 31 species-specific markers, and a list of genes expressed in CFSC-2G established by bulk mRNA next-generation sequencing (NGS). During the last years, immortalized HSC lines have become an established experimental tool in biomedical research in place of primary cells. Compared to primary cells, immortalized HSC lines offer several advantages. They are cost effective, easy to handle, and provide an unlimited supply of biological material. Moreover, they can bypass ethical concerns that may arise by isolation of primary cells from animal or human tissue . In addiPresently, continuous HSC lines were established from mouse, rat, pig, and humans. Although widely applied, only several of these continuous lines have been genetically characterized, while numerous scientific journals and funding agencies now request cell authentication before considering publication or funding when working with cell lines. Some years ago, we described genetic characteristics of the human HSC line LX-2 . In addiNevertheless, based on the fact that results obtained from one specific cell type might require the reproduction in another cell line, we here established genetic characteristics for the cirrhotic fat-storing cell line (CFSC). This cell line and four different clonal sublines derived thereof, i.e., CFSC-2G, CFSC-3H, CFSC-5H, and CFSC-8B, were originally established in the laboratory of Marcus Rojkind from a carbon chloride cirrhotic rat liver and proposed as a useful tool to study extracellular matrix production by HSCs ,8.In the present study, conventional chromosome analysis and multicolor spectral karyotyping were accomplished to gain insight into the genetic organization of CFSC-2G cells that has successfully uncovered numerous characteristic features of its chromosome organization. We further established a specific short tandem repeat (STR) profile for this cell line that includes 31 species-specific allelic variant sites. Bulk sequencing of mRNA isolated from CFSC-2G cells confirmed the expression of typical HSC markers associated with extracellular matrix synthesis and turnover. In addition, electron microcopy and Rhodamine–Phalloidin stain confirmed that CFSC-2G cells form a robust microfilament network, cytoplasmic fat droplets, and other cellular features typical for HSCs. In sum, our study provides genotypic and phenotypic characteristics of CFSC-2G cells that are useful for cell authentication.Papers using CFSC cells were identified by searching the PubMed database  using thCFSC cells were established and characterized over thirty years ago in the laboratory of Marcus Rojkind ,8. It is4)  solved in 25 mM sucrose buffer (Merck). Fixed cells were dehydrated and subsequently incubated in propylene oxide  in a mixture of Epon resin (Serva) and propylene oxide (1:1) and pure Epon. Finally, ultrathin sections (70–100 nm) were prepared and packed upon Cu/Rh grids . Contrast was enhanced by staining with 0.5% uranyl acetate and 1% lead citrate . The samples were analyzed at an acceleration voltage of 60 kV using a Zeiss Leo 906  transmission electron microscope and depicted images were taken at magnifications of 2156× to 35,970×.Electron microscopic analysis of CFSC-2G cells was essentially conducted as described before . In briew/v) hypotonic potassium chloride solution and fixed with a mixture of methanol and acetic acid (3:1). Air-dried chromosome spreads were prepared, and slides were treated with 0.025% (w/v) trypsin solution followed by staining with Giemsa solution to visualize G-banding pattern. In addition, the heterochromatin in metaphase chromosomes was stained following an established procedure ..23].Rattus norvegicus reference transcriptome. The complete list of mRNAs detected with their abundances is given in −11  to 12,768.5 . In particular, we found strong expression of α-smooth muscle actin , collagen type I α1 , collagen type III α1 , Vimentin , two Fibronectin 1 transcripts , galectin 1 , secreted protein acidic and cysteine rich , tissue inhibitor of metalloproteinases 1 , Timp2 (TPM: 1030.84), Cd63 (TPM: 2732.07), transgelin 2 , cofilin 1 , caveolin 1 , and cysteine and glycine-rich protein 2 , respectively. All these markers indicate that CFSC-2G cells originate from primary HSCs. The expression of most of these markers was recently demonstrated in rat HSC line HSC-T6 cultured under the same culture conditions (Bst2), representing a component of the innate immune response to enveloped viruses. Similar to HSC-T6, the NGS data demonstrated that CFSC-2G cells express large quantities of ferritin heavy chain 1 , ferritin light chain 1 , and ferritin light chain 1-like protein , which are associated with the intracellular storage of iron. Moreover, in line with their activated phenotype, CFSC-2G cells express only low mRNA amounts of the peroxisome proliferation-activated receptor-γ (Pparg), representing one nuclear receptor whose activity is significantly decreased in activated HSCs [We next isolated mRNA from CFSC-2G cells cultured under basal conditions to 80% confluence and performed bulk mRNA sequencing (mRNA-Seq). The identity and abundance of each mRNA were determined using the nditions . FurtherEnumeration of Giemsa-stained chromosomes of CFSC-2G cells at different intervals consistently showed a shift from the standard diploid chromosome number that could be characterized as a hyperdiploid karyotype with varying number of chromosomes ranging from 53 to 65 . No cellConsistent with the GTG-banded chromosomes, large bi-armed rearranged chromosomes were noted in the CBG-banded metaphase plates cf. A. ThoughStrikingly, cytogenetic analysis in CFSC-2G cells further detected the sporadic occurrence of ring chromosomes in metaphases that appeared to differ in size and were partly di-centric C–E. In tDue to the specific color code assigned to individual chromosomes, the SKY analysis precisely allows authentic identification of both structural and numerical aberrations. p-) material in all these RNO1 derivatives. Both RNO2 and RNO7 are organized in multiple copies, but these two autosomes displayed unusual organization for a bi-armed chromosome that may arise through isochromosome formation, pointing to an overt duplication of the long arms for RNO2 and RNO7. Additionally, the SKY analysis disclosed translocations of different chromosomal segments in the derivative chromosomes of RNO2, RNO4, RNO6, RNO10, RNO14, RNO17, and RNO19. Both RNO9 and RNO18 are the only ones that are organized in two copies. On the other hand, three chromosomes  are present in two copies, but the SKY analysis revealed that additional parts of these chromosomes were also found in other derivative chromosomes. Likewise, RNO15 and RNO16 occurred in single copy, but multiple segments from these two chromosomes were found in different derivative chromosomes.However, the most striking observation was found for chromosome 1 (RNO1), which was found in five copies. All RNO1 copies are literally derivative ones in which the tiny, short arms are replaced by different chromosome segments, hinting to chromosome translocations and simultaneous loss of the short arm  DNA profiling using a set of 31 established polymorphic markers. The resulting PCR products were analyzed on a genetic analyzer resulting in a well analyzable electropherogram for each marker tested  that all4 cirrhotic liver [Continuous HSC lines resemble primary cultured HSCs in many ways and are valuable and useful tools to address issues of HSC biology and function. As a result, the number of studies which use HSC lines is steadily increasing. However, from the ~30 HSC lines available, only a few lines have found widespread use . In regaic liver ,8. SinceNevertheless, the chromosomal and genetic characteristics of this cell line were not established. It is well accepted that continuous cell lines are under permanent stress and selection due to the artificial environment in which they exist. Genetic mutation can occur during cell passaging and laboratory manipulation, which can lead to a heterogeneous population with a selective advantage. As a consequence, this could result in a final cell population that is completely different from the starting pool. Unfortunately, the factors provoking a genetic drift within a cell line can be different in various labs.A fundamental study has shown that established cell lines, generally thought to be clonal, are highly genetically heterogeneous, most likely resulting from clonal dynamics and continuous instability . It is sConsequently, it is necessary to identify resilient biochemical and genetic features of a continuous growing cell line to confirm the identity and genetic stability and to mark the ‘biochemical quality’.In our study, we describe several molecular features of CFSC-2G cells that are useful for cell authentication. The cell line has a fibroblastic morphology that is in agreement with the proposed fibrogenic phenotype . ElectroFurthermore, CFSC-2G cells have a prominent network of intracellular actin filaments  and are Acta2, Col1a1, Col3a1, Vim, Fn1, Lgals1, Sparc, Csrp2, Timp1, Timp2, Tagln2, retinol-binding proteins, and their nuclear receptors , matrix metalloproteinase-2 (Mmp2), Timp1, Timp2, and plasminogen activator inhibitor-1 (PAI-1/Serpine1) in CFSC-2G [Previous work demonstrated that the treatment with all-trans retinoic acid (ATRA) inhibits proliferation and diminishes mRNA expression of procollagen α1(I), procollagen α1(III), transforming growth factor-β1, connective tissue growth factor -induced cirrhotic male Wistar rats [4 is a non-genotoxic carcinogen known to be associated with intra-chromosomal recombination provoking liver cancer in rats and mice [4. The observation of multiple copies of chromosomes leading to a pseudo-triploid karyotype can be considered as a significant and specific feature of CFSC-2G cells, since similar multi-copy chromosomes are absent, for example, in the rat HSC-T6 cell line [It is noteworthy to mention that the recent analysis of the rat hepatic stellate cell line HSC-T6 that was established through SV40T transfection surprisingly showed relatively less chromosome changes without significant change in the modal chromosome number [tar rats ,8. CCl4 and mice . Therefoell line ,30. Inteell line ,32,33. Mell line .Essentially, the karyotype abnormality described in this study provides insight to the genomic instability of CFSC-2G. The numerical and sweeping chromosomal changes might contribute to the capacity of this cell line to proliferate indefinitely.Altogether, chromosome banding and multicolor SKY analysis revealed that CFSC-2G cells are characterized by extensive aneuploidy and striking chromosomal rearrangements that might be too complex to be used in daily routine for cell authentication. Therefore, we next performed STR DNA profile analysis involving the simultaneous amplification of 31 rat-specific STR markers. This analytical DNA technique is suitable to establish a characteristic fingerprint of these hypervariable microsatellite regions, allowing unambiguous cell authentication. The analysis revealed a unique STR profile in which 18 markers produced only one peak in the analysis and can be considered homozygous, while 13 markers gave rise to two peaks and can therefore be considered to be homozygous . The finIn sum, the presented biochemical characteristics, standard karyotyping analysis, SKY analysis, and the STR profile established in this study are certainly suitable means that can be used for cell authentication of CFSC-2G cells. In the future, this information will increase the biomedical value of CFSC-2G cells in liver research. In addition, our study helpful to fulfill the requirement of increasing the number of scientific journals and funding agencies that have started to require cell authentication prior to paper or grant submission when working with cell lines.CFSC-2G cells are an attractive cellular model to analyze aspects of HSC biology. Since the original establishment of these cells three decades ago in the Marion Bessin Liver Research Center , the cells have made their way into many laboratories worldwide. The identification of specific chromosomal alterations and the definition of a unique STR profile will now allow discriminating these cells from all other cell lines. The demonstration that CFSC-2G cells express typical markers of HSCs , have a robust microfilament network, and can uptake and store fats confirms the proposed activated HSC phenotype. Altogether, the identification of a highly characteristic genetic profile will further increase the biomedical value of CFSC-2G for liver research. Together with cell line HSC-T6 that was recently genetically characterized by our team, researchers have now two independent, genetically well-characterized cell lines in hand that can be mutually used to reproduce experimental findings in continuous growing HSC lines of rat origin.