Ovarian toxicity is most common gynecologically related malignancy, arising for most cases owing to the advanced stage of diagnosis. The aim of the current study was to explore the anticancer potential of baicalin against cyclophosphamide (CP) induced ovarian toxicity in mice and explore the possible mechanism. ovarian cancer cells  were treated with different doses of baicalin and examined via flow cytometry and cell proliferation assay. Subcutaneous administration of CP (200 mg/kg) was used to induce the ovary toxicity and mice were received the oral administration of baicalin. Oxidative, pro-inflammatory, inflammatory, apoptosis parameters, progesterone, estrogen hormones and histopathological were also estimated at end of the study. Baicalin increased the apoptosis and caused the cell cycle arrest at the G2/M stage in ovarian cancer cells. Baicalin significantly (P < 0.001) reduced the level of TGF-β in the HO8910PM, SKOv3ip and Hey cell lines. Baicalin significantly (P < 0.001) increased the body weight and reduced the tumor volume in mice. Baicalin significantly (P < 0.001) increased the level of estrogen and progesterone. Baicalin significantly (P < 0.001) reduced the level of malonaldehyde (MDA) and increased the level of superoxide dismutase (SOD) and catalase (CAT). Baicalin significantly (P < 0.001) decreased the level of pro-inflammatory cytokines such as tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), interleukin-6 (IL-6) and inflammatory parameter such as nuclear kappa B factor (NF-κB), respectively. Baicalin significantly (P < 0.001) reduced the level of the caspase-3. Baicalin, act as the potential agent against the ovarian toxicity by alteration of TGF-β and inflammatory pathways. OvariIt is well proofed that the cyclophosphamide (CP) is an alkalyting agent and commonly used to treat the various types of cancer. The toxic and therapeutic effects of CP depend on related active metabolites as phosphoramide and acrolein ,11. DuriThe current approach for cancer prevention involves the use of plant-based drugs, isolated compounds from plants, dietary products, or synthetic compounds that serve to prevent or suppress the progression of malignancy. This approach has become increasingly popular due to the rising global incidence of cancer . Some of22.1The human ovarian cell lines Hey and SKOv3ip were obtained from the Anderson Cancer Center . RPMI or DMEM medium  was used for the culture the cell lines. The ovarian epithelial surface was lightly scraped to yield non-cancerous ovarian surface epithelial (OSE) cells, which were then cultured in medium 199:105 supplemented with 15 % foetal bovine serum and 10 ng/ml EGF , as previously described . The Nan2.2For the estimation of cell viability effect, we performed the methylthiazoltetrazolium (MTT) assay . Each we2.3The ovarian cancer cell lines and OSE cells were seeded on the 96-well plates and incubated for 3 days with different baicalin concentrations (0–240 μM for OSE cells and 0–40 μM for cancer cell lines). Added 50 μl trichloroacetic acid (30%) and incubated again for 1 h at 4 °C. The cell lines were washed and dry, 100 μl solution of SRB (0.4 %) was added for ½ h. the acetic acid (01 %) was used for the washed the plate and air dried, after that Tris base (100 μl) was added, and the prepared plate was shaken for 5 min and the SRB value was estimated at the 490 nm wavelength ,22.2.4Hey and SKOv3ip cellline were harvested in the 60 mm plates and incubated with baicalin (20–40 μM) and DMSO from the next day. The tested and control cells were trypsinized after the 24–48h treatment, collected in PBS and fixed on the ice, followed via raising with cold ethanol (70 %). After the treatment, the cells were stained with propidium iodide (50 μg/ml) at room temperature for 15 min. Flow cytometry was used for the estimation of cell cycle analysis of stained cells .2.5Ovarian cancer cell lines were harvested in the plate (60 mm) and incubated with baicalin or DMSO. After 1, 2 and 3 days the culture supernatants were seed and the concentration of TGF-β1 was estimated. 96 well plates were used for harvested the Hey cells and incubated with the baicalin, or DMSO (Control) and Paclitaxel. SRB was used for the determination of the protective effect of baicalin on the proliferation of the ovarian cancer cell lines. ELISA reader was used for the estimation the concentration of TGF-β1 via using the available manufacturer's instruction.2.6Swiss albino mice (20 ± 5 g) sex-female was used in the current experimental study. The mice were received from the departmental animal house and kept in the standard laboratory condition . All the animal study was carried out according to the institutional animal protocol. The whole animal study was approved from the Xi'an International Medical Center Hospital .2.7The cyclophosphamide (toxicant drug) was used for the induction of ovarian toxicity. Subcutaneous administration of CP (200 mg/kg) was used for the induction of ovary toxicity . The tes2.8The mice were divided into different groups as follow and each group contains the 12 mice in each groups. Group I: normal control; Group II: cyclophosphamide (CP) control; Group III-IV: CP control treated with baicalin  [2.9Progesterone and estrogen level in the serum was estimated via using the ELISA kits following the manufacture instructions . The quantity of progesterone and estrogen in the samples were estimated via using the standard curves and the amount was expressed in ng/l.2.10The antioxidant enzymes such as superoxide dismutase (SOD), catalase (CAT) and malonaldehyde (MDA) were estimated using the ELISA kits following the manufacture instruction .2.11Cytokines parameters like tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), interleukin-1β (IL-1β), interlukin-10 (IL-10) and inflammatory parameters such as nuclear kappa B factor (NF-κB) were estimated using the  following the manufacture instruction.2.12Caspase parameters were determined using the ELISA kit  following the manufacture instruction.2.13All the presented data were subject to statistical analysis and shown as the mean ± standard deviation. Two-tailed t-tests and data counting were evaluated using the chi-square criteria that measures parameter frequency.33.1The effect of baicalin on cell viability was evaluated on different ovarian cancer cell lines, including HO8910PM a, SKOv3i3.2For the estimation of mechanism, we estimated the apoptosis effect of baicalin on the different ovarian cancer cell lines (data not showed). Baicalin treatment exhibited the reduced CXCR4 mRNA expression on the different cancer cell lines. Baicalin acid also decreased the protein level of CXCR4 and could be activated through SDF-1a in primary ovarian tumors and ovarian neoplasms .3.3For the estimation of baicalin capacity to bind the extracellular matrix, we executed the cell adhesion assay using the ovarian cancer cell lines . 3.43.5For the estimation of cancer cell migration, in vitro scratch assay performed. All the cells were pre-incubated in the FBS medium for 24 h before the cell proliferation. The wound healing effect was supervised at regular intervals till its completed closed. 3.6Baicalin was used for the estimation of inhibitory effect against the inflammatory mediators. Previously research suggests that the high concentration of TGF-β1 was observed in the cancer patient. 3.73.83.9CP induced ovarian toxicity exhibited the increased level of pro-inflammatory cytokines such as TNF-α a, IL-6 b, IL-1β 3.103.11During the ovarian toxicity, the level of estrogen a and pro3.124For chemotherapy, cyclophosphamide is the primary choice for the treatment of ovarian cancer. However, it is associated with serious side effects, and the development of resistance to cyclophosphamide poses a significant obstacle ,27. AnotIn vitro ovarian cancer activity showed that the baicalin induced the cell cycle to seize at the G2/M stage and also increased the apoptosis [p-cdc2 in all cancer cell lines, which might exhibit the molecular level effect via inducing the cell arrest at G2/M phase. Its also confirmed reduced effect of p-Wee1 and Myt1 on all cell lines, showed the efficiency on baicalin in inducing the cell capture on G2/M phase. This effect is occurred due to inhibit the cdc2 and Cyclin B1 via Wee1 and Myt regulation.poptosis . We alsopoptosis ,32. Cyclpoptosis ,34. In tCP causes the DNA damage in cancer cells and its enhance the survival of cancer patients, but also induce the side effect on the female reproductive system including temporary or permanent amenorrhea decreased infertility or fertility ,11. It iSeveral researchers suggest that the TGF-β1 play an imperative role in the epithelial-mesenchymal transimition (EMT) and metastasis of ovarian cancer cells . The res5in-vivo and in-vitro), although it showed the low toxic effect against the normal cells. Baicalin reduced the secretion of TGF-β1 and also blocked the Snail stabilization that is caused by the TGF-β1. Baicalin also blocked the non-canonical ERK/AKT pathway and canonical Smad activation. Baicalin, by demonstrating the activity on the TGF-β/AKT/ERK/Smad signaling pathways (Our experimental study showed that the baicalein significantly reduced the progression of ovarian cancer cells (pathways , serve aThis work was supported by Xi'an International Medical Center Hospital.Data will be made available on request.The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.


Drosophila neuroblasts, Rho1 is controlled by two Pbl isoforms with distinct localisation. Spindle midzone- and furrow-enriched Pbl-A focuses Rho1 at the furrow to sustain efficient ingression, while Pbl-B pan-plasma membrane localization promotes the broadening of Rho1 activity and the subsequent enrichment of myosin on the entire cortex. This enlarged zone of Rho1 activity is critical to adjust furrow position, thereby preserving correct daughter cell size asymmetry. Our work highlights how the use of isoforms with distinct localisation makes an essential process more robust.Cytokinesis partitions cellular content between daughter cells. It relies on the formation of an acto-myosin contractile ring, whose constriction induces the ingression of the cleavage furrow between the segregated chromatids. Rho1 GTPase and its RhoGEF (Pbl) are essential for this process. However, how Rho1 is regulated to sustain furrow ingression while maintaining correct furrow position remains poorly defined. Here, we show that during asymmetric division of  This study provides evidence that two RhoGEF isoforms displaying distinct localisation concurrently modulate Rho1 activity to promote robust furrow ingression while preserving cell size asymmetry during neural stem cell division. The activity of the small GTPase RhoA (Rho1 in Drosophila) is essential for this process as it promotes actin nucleation and myosin activation6. Rho1 is a small GTPase of the Ras super-family that undergoes nucleotide-induced conformational changes as it toggles between inactive GDP- and active GTP-bound states. Rho1 localises to the plasma membrane through prenylation at its carboxy-terminus and its lipid interaction is obligatory for function7. The zone of active Rho1 at the plasma membrane determines the position of contractile ring assembly and subsequent cleavage furrow ingression9. Rho1 activation is catalysed by a guanine nucleotide exchange factor (GEF) called Ect2  in mammals13 and Pebble (Pbl) in Drosophila15. Hence, the mechanism controlling Ect2/Pbl localisation and activation is critical to determine the zone of Rho1 activation and thus the position of the resulting furrow. Ect2/Pbl contains BRCT  domains in its N-terminus and a catalytic DH (Dbl homology) domain and juxtaposed membrane-associated PH (Plekstrin homology) domain in its C-terminus. The canonical mechanism by which Ect2/Pbl concentrates at the equatorial zone to activate Rho1 is through its interaction with mgcRacGAP/Cyk4 (RacGAP50C or tumbleweed in Drosophila) via the BRCTs18. RacGAP50C forms part of the conserved centralspindlin complex with the kinesin 6 MKLP1/Zen4 (Pavarotti in Drosophila). This complex promotes the bundling of anti-parallel microtubules at their plus-end to form the midzone of the central spindle that assembles between the two segregated pools of chromatids20. In vertebrates, the phosphorylation of mgcRacGAP by Polo-like Kinase 1 provides docking sites for Ect2’s BRCT domains, thereby relieving Ect2 auto-inhibition and promoting its accumulation at the spindle midzone and equatorial membrane24.Cell cleavage, also called cytokinesis, is a fundamental process that occurs subsequent to sister chromatid segregation. In animal cells, cytokinesis relies on the assembly of a contractile ring composed of filamentous actin and bipolar filaments of non-muscle myosin II (referred to as myosin) anchored at the equatorial plasma membrane. Myosin activity generates the force necessary to drive the ingression of the cleavage furrow. The topological resolution of the two resulting cells is achieved by abscission of the membraneC. elegans Q neuroblast lineage, where the smaller daughter cells undergoes apoptosis25.While the molecular pathway that specifies the zone of Rho1 activation to initiate contractile ring assembly is well defined, less is known about the mechanisms that maintain the contractile ring position while sustaining efficient constriction. The study of these mechanisms is hindered by the fact that severe perturbation of either Rho1 signalling or the kinetics of contractile ring components during division results in failure to initiate furrowing or furrow regression. However, the mechanism that controls Rho1 activity to sustain furrowing at the right position is critical to preserve the size of the resulting daughter cells. This is particularly important during asymmetric stem cell division where cell size determines cell fate, as observed in the Drosophila larval neuroblast is a powerful model for studying the mechanisms that spatio-temporally control the asymmetric position of the furrow. The division of this large stem cell produces two cells that differ in size and fate: the larger cell retains the neuroblast “stemness” and continues dividing frequently, while the smaller ganglion mother cell (GMC) undergoes one round of division before differentiating into neuronal lineages26. Previous studies have shown that the asymmetric position of the furrow is specified by two pathways, a spindle-independent pathway that requires the polarity complex Pins (composed of Partner of Inscuteable (Pins); Gαi; Discs Large) to bias Myosin activity towards the basal cortex and a microtubules-dependent pathway that regulates RacGAP50C localisation to the equatorial cortex35.The 36. This pool of active myosin at the poles becomes critical if chromatids trail at the cleavage site during furrow ingression. Under these conditions, cell elongation ensues from the reorganisation of polar myosin into broad lateral rings that partially constrict. This “adaptive” cell elongation facilitates the clearance of trailing chromatids from the cleavage site. Attenuation of Pbl function impairs myosin polar cortex dynamics during division. Consequently, cells cannot undergo adaptive elongation in the presence of trailing chromatids, with dire consequences for tissue homeostasis37. While these Pbl-dependent changes in the mechanical properties of the polar cortex are important for the clearance of trailing chromatids, little is known about their role in maintaining the fidelity of asymmetric neuroblast division under normal conditions.In addition to these pathways, our team recently identified novel Pbl-dependent myosin dynamics during neuroblast division. At mid-ring closure, a pool of myosin undergoes outward flow (efflux) from the contractile ring, invading the entire cortex  until the end of furrow ingressionpblMS mutation, an hypomorph allele of pbl, defects in daughter cell size asymmetry arise during division. We identified pblMS as a non-sense substitution in an alternatively spliced exon. The mutation prevents the production of Pbl-B, one of two major Pbl isoforms that we identify. However, the pblMS mutation does not affect Pbl-A, the sole Pbl isoform reported in the literature thus far. We found that unlike Pbl-A, Pbl-B is not enriched at the central spindle midzone and furrow. Additionally, it is sequestered in the nucleus prior to Pbl-A. Genomic rescue experiments revealed that the lack of a functional Pbl-B recapitulates pblMS cytokinesis phenotypes including the lack of myosin polar cortex enrichment and the formation of abnormally small GMC. In contrast, the lack of Pbl-A expression induced excessive myosin enrichment to the polar cortex associated with a transient but dramatic widening of the furrow that mildly affected its rate of ingression. These phenotypes are similar to those observed after depletion of RacGAP50C. We propose that two Pbl isoforms displaying distinct localisation patterns modulate Rho1 activity concurrently to adjust furrow position during ingression and maintain cell size asymmetry.In this study, we investigate the consequences of perturbed myosin polar cortex enrichment due to reduced Pbl activity during asymmetric cell division by live imaging of neuroblasts. We found that in the absence of myosin polar cortex activity associated with the Drosophila larval neuroblast cytokinesis in wild type (WT) and a homozygote mutant for an hypomorph allele of pbl called pblMS. In WT neuroblasts, myosin adopted a uniform cortical localisation at anaphase onset (initiation of sister chromatid separation) , a pool of myosin underwent outward flow from the ring and enriched the entire cortex of both nascent daughter cells Fig. . No diffpblMS. To do so we first measured the area of the resulting GMC at the end of ring closure. Remarkably, a significant proportion of pblMS cell divisions led to the production of abnormally small GMCs, suggesting a defect in furrow position during ingression  with the area of the resulting GMC. Strikingly, in contrast to WT cells, a strong correlation was found between the initial position of the furrow and the size of the resulting GMC in pblMS  from pblMS male adult genomic DNA extracts and compared these with the pbl sequence of the wild type strain we had in the lab. The Flybase database annotation reports that the pbl gene is encoded by 15 exons spread over 15 kb on chromosome 3 L (cytologic position 66A18-66A9 and genomic position 7896109-7911954). We identified a cytosine to thymidine substitution in exon 8 at position 7901087 on chromosome 3 L in pblMS DNA extracts from three independent experiments. This substitution changes the glutamine codon CAG into the stop codon TAG   Fig. . Hence, pbl genomic sequence tagged with GFP 43. This is consistent with a recent phylogeny study on the functional evolution of BRCT domains, which identified a third BRCT in the N-terminus of Ect244. The structural analysis of the N-terminus of Ect2 confirmed the presence of three apposed BRCT domains45. While there is evidence that two BRCT domains are involved in Pbl and human Ect2 binding with RacGAP50C/MgcRacGAP, less is known about the role of the third BRCT (BRCT2) in this interaction45. We found that this sequence is inserted in the middle of the 76 amino acid region that is predicted to form the BRCT2 domain in Pbl-B   in addition to the conserved N-terminal BRCT1 and 2 in Pbl-A Fig.   Fig. 43. Sincepbl promoter. We monitored GFP::Pbl-A or GFP::Pbl-B signals during cytokinesis in live neuroblasts and compared their localisation with VenusFP::RacGAP50C, the centralspindlin component known to interact with Pbl-A . Collectively, these results indicate that the presence of exon 8 promotes premature Pbl-B nuclear localisation and either inhibits its midzone localisation or favours its broad membrane localisation and premature nuclear localisation at the expense of the midzone.Next, we investigated Pbl-B and Pbl-A sub-cellular localisation during cytokinesis. To do so, we produced transgenic fly strains expressing GFP::Pbl-A or B under the control of the endogenous l-A Fig. 19. Recenane Fig. . Intriguane Fig. . Upon coane Fig. . GFP::Pbane Fig. 36. Whilease Fig. . ImportapblMS cells that lack a functional Pbl-B suggest that the two isoforms act differently to control Rho1 activity during cytokinesis. To examine the role of Pbl-A and B isoforms individually during neuroblast division, we adopted a genomic rescue approach. We took advantage of an existing pAcman plasmid containing a 17 kb sequence including the pbl genomic sequence and the corresponding transgenic flies containing the pbl full-length construct (referred hereafter as Pbl-A + B)46. We produced two modified versions of this pAcman plasmid and the corresponding transgenic flies where the constructs were integrated at the same locus as the full-length to minimise variation in transgene expression. In the first version, we introduced a non-sense substitution in exon 8 mimicking the pblMS mutation, and thus preventing the production of a functional Pbl-B (referred hereafter as Pbl-A). In the second version, the intron between exon 8 and 9 was deleted to prevent alternative splicing of exon 8 and thus promote the expression of Pbl-B but not Pbl-A (referred hereafter as Pbl-B)  Fig. . All thrced Fig. . Next, wced Fig. . Pbl-B lpbl constructs and the myosin marker Sqh::GFP. pbl3/pbl2 neuroblasts expressing both Pbl-A and B (Pbl-A + B) exhibited myosin dynamics similar to WT cells, including myosin polar cortex enrichment at mid ingression  pathway49, the pblMS phenotypes observed during neuroblast divisions are likely the result of a loss of Pbl-B function rather than the dominant effect of truncated Pbl-B expression. However, we cannot formally exclude the latter possibility.The identification of the 16 and (2) its Rho1-independent role in the epithelial to mesenchymal transition (EMT) and mesoderm cell migration54. The fact that the expression of Pbl-B rescues the embryonic lethality of pbl null mutants and produces adult escapers indicates that it contributes to some extent redundantly with Pbl-A to perform these two independent functions.No role has been assigned to the Pbl-B isoform prior to our work. Two distinct functions for Pbl during embryonic development have been identified through genetic screens: (1) its function as a GEF for Rho1 during cytokinesisC. elegans one-cell embryos revealed that two parallel pathways, a Cyk-4-dependent and independent pathway, drive furrow ingression56. In mammalian cells, the association of Ect2 with the plasma membrane or the local activation of RhoA through the use of optogenetics is sufficient to induce furrowing57. Remarkably, successful cytokinesis occurs in cells expressing Ect2 mutated on residues that impairs its interaction with MgcRacGAP and prevents its recruitment to the spindle midzone, highlighting an MgcRacGAP-independent redundant pathway for Ect2 activation57. Recently, a novel isoform of human Ect2 has been identified through screens for human transformed cells resistant to treatment with Doxorubicin and Paclitaxel59. Importantly, this long human Ect2 isoform is strictly nuclear in interphase like Pbl-B and, also reminiscent of Pbl-B, contains additional amino acids in one of the BRCT domains that may affect its folding and impair Ect2 interaction with MgcRacGAP. Thus, the use of redundant RacGAP-dependent and independent pathways for Ect2/Pbl activation during cytokinesis may be a convergent mechanism in multicellular organisms, making a process that has to adapt to a variety of cell types with specific extrinsic and intrinsic physical constraints more robust.Structural modelling predicts that Pbl-B, like Pbl-A, may contain three folded BRCTs at its N terminus despite the presence of 458 extra amino acids within the third BRCT sequence. Since BRCTs are critical for Pbl-A interaction with RacGAP50C, this implies that Pbl-B may bind RacGAP50C. However, several lines of evidence suggest that, in vivo, Pbl-B functions independently of its interaction with RacGAP50C. First, Pbl-B is not detected at the spindle midzone and is not concentrated at the furrow, whereas Pbl-A localises to the midzone and furrow site in a RacGAP50C-dependent manner. Second, expression of only the Pbl-B isoform produces phenotypes, including an enlarged furrow and slower rate of ingression, akin to that observed in cells depleted for RacGAP50C. Hence, we propose that two parallel pathways, a RacGAP50C-dependent Pbl-A and a RacGAP50C-independent Pbl-B pathway act in concert to promote robust asymmetric division in neuroblasts. Consistently, studies in Drosophila neuroblasts. One pathway involving the Pins polarity determinant complex acts early to promote myosin depletion from the apical pole and accumulation at the basal cortex33. The second pathway depends on the microtubules-associated centralspindlin complex that acts to stabilise the baso-equatorial position of the contractile ring31. Since myosin dynamics during furrow site specification are unperturbed in cells lacking Pbl-A or depleted for RacGAP50C, we propose that the polarity-determinant pathway may be the predominant pathway that positions the cleavage site in these cells. The target of the polarity pathway for cleavage site specification is currently unknown. It would be interesting in future to determine if this pathway acts through Pbl-B regulation.The model that Pbl-B drives cytokinesis independently of RacGAP50C raises the question of how the furrow site is specified in these cells. Previous studies have identified two distinct pathways that define the future cleavage site in 31. In this study, perturbation of the peripheral microtubule-associated pool of centralspindlin produced larger GMCs than normal. Since our data suggest that Pbl-B acts independently of the centralspindlin complex and that cells lacking Pbl-B produce GMCs that are smaller than normal, we propose that an additional mechanism to the centralspindlin-dependent pathway participates in furrow position adjustment. This mechanism is likely to involve the change in mechanical properties of the nascent daughter cell cortices as active myosin enriches the cortex during ring constriction. Indeed, myosin enrichment at the polar cortex coincides with an increase in contractility, as illustrated by the transient deformation of the nascent cells  competitively influence furrow position during ingressionlls Fig. . SupportWe provide the ground-work for future studies on how changes in the mechanical properties of the polar cortex contribute to adjust furrow position during asymmetric stem cell divisions. It will be equally interesting to assess this question in other cell types and in relation to tissue features. Pbl-A and B isoforms provide excellent genetic tools to address these questions as they allow the physiological modulation of Rho1 activity at the furrow and polar membrane.pbl2 and pbl3 alleles were described previously47. The screen that identified pblMS  was described in ref. 38. P[UAS > RacGAP50C-dsRNA] (stock n°6439) and P[69B > Gal4] strains were provided by Bloomington stock centre . P was described in ref. 18. Pbl-A + B stock was described in ref. 46. Pbl-A, Pbl-B, GFP::Pbl-A and GFP::Pbl-B transgenic stock were produced in our laboratory for this study  was added and the mixture incubated on ice for 10 min. After centrifugation at 12,000 g for 15 min, 1 mL of supernatant was transferred to 600 µL of isopropanol. After 15 min of centrifugation at 12,000 g, the pellet containing the genomic DNA was washed with 70% ethanol and dried before resuspension in 150 µL of diH2O.pblMS flies  and adult testes, total RNAs from 60 larval CNS or 80 adult testes were extracted using RNAspin Mini kit (GE Heathcare). 1 µg of total RNAs of each extract were converted into cDNA using oligodT and SuperScript III Reverse transcriptase (Invitrogen) according to the manufacturer’s protocol. Quantitative PCR was performed using SSO Advanced Universal SYBR Green Supermix (Bio-Rad).The identification of 46. For Pbl-A, the cytosine at position 1114 (Q372) was substituted to thymidine to mimic the pblMS mutation. For Pbl-B, the intron sequence between exons 8 and 9 was removed (Genebridges). GFP::Pbl-A and GFP::Pbl-B were cloned as follows: Pbl-A and B cDNAs produced by RT-PCR from adult mRNA extracts were inserted into pAcman[GFP] using AscI and NotI sites flanking a cassette obtained by PCR and containing sequences as follows: AscI-pbl promoter-pbl5’UTR-GFP-pbl (A or B) cDNA -pbl3’UTR-NotI. The resulting plasmids pAcman[Pbl-A], pAcman[Pbl-B], pAcman[GFP::Pbl-A] and pAcman[GFP::Pbl-B] were used to produce transgenic flies with the insertion at the same landing site, 86F8b on the third chromosome. The plasmid pAcman[GFP::Pbl-A + B] was produced by inserting the eGFP sequence before the ATG of the genomic Pbl sequence present in pAcman[Pbl] using AscI and BsiW restriction enzymes. The resulting pAcman[GFP::Pbl] plasmid was inserted at the landing site 68A4 on the third chromosome of the fly genome.Pbl-A and B constructs were obtained by DNA synthesis using pAcman[Pbl] plasmid described previously60 brains from 3rd instar larvae of each genotype were dissected in  1X PBS and transferred in 30 µL of RIPA buffer  supplemented with protease inhibitor cocktail (Roche) and PMSF (1 mM). After grinding and homogenization with a pestle in a 1.5 mL centrifuge tube, lysates were centrifuged at 15,000 g for 5 min at 4 °C and supernatants containing proteins were transferred into equal volume of Laemmli 2X buffer. Boiled samples were loaded and analysed by 10% SDS-PAGE. Proteins were transferred to nitocellulose membrane using a semi-dry electrophoretic blotting device (BioRad). Mouse anti-GFP  and mouse anti-αTubulin primary antibodies  and anti-mouse HRP conjugated secondary antibodies  were used for western blotting.66. The preparation was kept for a maximum of one hour. In this study, neuroblasts above 11 µm in diameter were observed except for Fig. Live imaging of larval neuroblasts was carried out as follows: late third instar larval CNS were dissected in 1x PBS and transferred to a drop of 1x PBS on a 22 × 22 mm glass coverslip. A glass slide (60 × 24 mm) was gently placed on top of the coverslip until the coverslip adhered to the slide by capillary action. The CNS was slightly squashed by absorption of excess liquid from the edges of the coverslip to facilitate visualisation of dissociated neuroblasts. The coverslip was then sealed with halocarbon oil 700 (Sigma)67. The background level of the camera was subtracted from all raw data before quantification. All fluorescence quantification was performed on a single plane of the relevant Z position. For the line-scan in Figs. cortex) and the cytoplasm (Myocyto) was measured using a 2-pixel wide line as illustrated in the schemes / Myocyto. A similar method was used to measure GFP::Pbl-A and B polar membrane and furrow enrichment shown in Fig. furrow) was measured by drawing a 2-pixel wide line at the furrow as indicated in the scheme in Fig. cortex/Myofurrow was then calculated. For Fig. Fiji  was used for image analysis and fluorescence quantificationes Figs. d and 4e.pbl2/TM6B were crossed with ten young females of the following genotypes: pbl3, pAcman[Pbl-A + B]/TM6B, pbl3, pAcman[Pbl-A]/TM6B, pbl3, pAcman[Pbl-B]/TM6B or pbl3/TM6B. For each genotype, the parents were left to lay eggs in a vial every 2 days for 8 days. The first vial was discarded and all the adult progeny of the remaining vials were counted. The percentage of viability of the transheterozygous individuals carrying the pAcman Pbl constructs was determined by calculating the ratio of pbl transheterozygote vs TM6B flies over the expected frequency of transheterozygotes according to Mendel’s law of inheritance. The experiment was performed three times independently.For viability assays, flies were raised on standard media at 25 °C. Eight young males 68. To quantify diploid and polyploid mitotic cells, preparations were observed by phase contrast using a Nikon eclipse Ti equipped with 100X objective.Third instar larval brain were dissected in 1X PBS, fixed 10 s in 45% followed by 10 s in 60% acetic acid solutions and squashed between slide and coverslip in an aceto-orcein solution to stain the chromosomeshttps://www.orthodb.org/?query=LOC4812378;42) and aligned with the sequence of the Pbl-B isoform of D. melanogaster. Sequences were aligned using MAFTT 7.40769 and run in “linsi” mode with options “–amino–reorder–treeout–localpair–maxiterate 1000”. The alignment was visualised in AliView 1.2670, ClustalX 2.171 and Seaview 5.0.572. This file is available as supplementary file “pbl_OrthoDB_eukaryota_MAFFT_alignment_reformatted.fasta.gz” in FASTA format.948 protein sequences of Pbl orthologs were retrieved from the OrthoDB v11 database (Further information on research design is available in the Supplementary InformationPeer Review FileDescription of Additional Supplementary FilesSupplementary Movie 1Supplementary Movie 2Supplementary Movie 3Supplementary Movie 4Reporting Summary


Introduction: Tyrosine kinase inhibitor drugs (TKIs) are highly effective cancer drugs, yet many TKIs are associated with various forms of cardiotoxicity. The mechanisms underlying these drug-induced adverse events remain poorly understood. We studied mechanisms of TKI-induced cardiotoxicity by integrating several complementary approaches, including comprehensive transcriptomics, mechanistic mathematical modeling, and physiological assays in cultured human cardiac myocytes.Methods: Induced pluripotent stem cells (iPSCs) from two healthy donors were differentiated into cardiac myocytes (iPSC-CMs), and cells were treated with a panel of 26 FDA-approved TKIs. Drug-induced changes in gene expression were quantified using mRNA-seq, changes in gene expression were integrated into a mechanistic mathematical model of electrophysiology and contraction, and simulation results were used to predict physiological outcomes.Results: Experimental recordings of action potentials, intracellular calcium, and contraction in iPSC-CMs demonstrated that modeling predictions were accurate, with 81% of modeling predictions across the two cell lines confirmed experimentally. Surprisingly, simulations of how TKI-treated iPSC-CMs would respond to an additional arrhythmogenic insult, namely, hypokalemia, predicted dramatic differences between cell lines in how drugs affected arrhythmia susceptibility, and these predictions were confirmed experimentally. Computational analysis revealed that differences between cell lines in the upregulation or downregulation of particular ion channels could explain how TKI-treated cells responded differently to hypokalemia.Discussion: Overall, the study identifies transcriptional mechanisms underlying cardiotoxicity caused by TKIs, and illustrates a novel approach for integrating transcriptomics with mechanistic mathematical models to generate experimentally testable, individual-specific predictions of adverse event risk. In contrast to conventional chemotherapeutics, these drugs are targeted treatments designed to inhibit particular protein kinases that are abnormally active in specific cancers. TKIs, of which over 40 have been approved by the US Food and Drug Administration , effectis by 20% . Unfortus by 20% . Despites by 20% , mechaniin vitro cell culture assays, such as cardiomyocytes derived from human induced pluripotent stem cells (iPSC-CMs). This approach has gained considerable momentum recently, and several recent studies have obtained important insight into TKI-induced cardiotoxicity  from two healthy female volunteers were differentiated into ventricular-like iPSC-CMs. These iPSCs were from a library generated in a previous study , which o2+], contraction, and vulnerability to proarrhythmic insults. Simulation results were used to prioritize experimental tests, and physiological optical recordings were performed to test key predictions. Experimental methods describing cell generation and drug treatments are described in Supplemental Methods; approaches that were developed for this study, such as integration of mRNAseq data with mathematical models, application of secondary insults, and experimental prioritization, are described here.After differentiating the iPSCs into iPSC-CMs, cells were treated with TKIs at Day 30 after differentiation initiation, and comprehensive transcriptomic screening was performed using mRNAseq to quantify how 48 h treatment with 26 FDA-approved drugs influenced gene expression. Methods for differentiation of iPSC-CMs and assessment of cell purity, summarized briefly here, are described in detail in Supplementary Materials and relevant citations. In brief, iPSCs were differentiated using an established protocol  that incTo improve purity for downstream assays, we used a metabolic selection protocol  that expThe mRNAseq libraries were prepared using a 3′-Digital Gene Expression (DGE) method . This me2+ handling (2+] levels in iPSC-CMs compared with adult ventricular myocytes (see Supplementary Methods).The mathematical model utilized in this study was constructed by integrating a model of human iPSC-CM electrophysiology and Cahandling  with a chandling  such thahandling . ParametTo simulate how TKIs may influence iPSC-CM physiology, we extracted from mRNA-seq datasets the genes that correspond to parameters in the mathematical model . The undEach new conductance was used to compute the relevant ionic current, with model equations otherwise unmodified.Contractile gene integration was performed using troponin C, actin, and myosin heavy chain (MHC) expression. Troponin C gene expression was integrated in an identical manner to ion channels; integration of actin and MHC involved more steps, as described in Supplemental Methods.2+ current, 2) block of delayed rectifier K+ current (IKr), and 3) hypokalemia. For hypokalemia simulations, we initially verified that K+ currents in the Paci et al. model of iPSC-CM electrophysiology  an increase in L-type Caysiology  respondemyocytes . Having K1, Ito, IKr, IKs, ICaL, INaK, INa, INCX). Over a single action potential, we integrated the total charge through each current and calculated how drug -induced gene expression changes affected the total amount of charge through that current, compared with untreated cells. At each level of secondary insult, this was expressed as ΔQ, the change in integrated current in the drug-treated compared with the untreated state. For INCX, which reverses during the action potential, we computed ΔQ for outward and inward currents separately  and the total number of cover slips (N) are reported for each condition.Recordings were first made during steady-state 1 Hz pacing with 5.4 mM extracellular , and membrane movement (2+] decay (Decay Tau) and CaT triangulation (CaD90/CaD50). These metrics are associated, respectively, with the reduced SERCA activity that occurs in heart failure . Some TKIs, such as nilotinib, were predicted to produce contractile failure through a combination of reduced CaT AUC and downregulation of contractile genes. In contrast, simulations predicted much smaller changes in CaT AUC resulting from regorafinib in Cell Line A or pazopinib in Cell Line B, and experiments failed to observe significant changes in this metric. In these cases, contractile failure resulted from dramatic downregulation of genes such as MYH7 and ACTC1.A striking finding in +]; 2) an increase in the magnitude of L-type Ca2+ current; and 3) block of the rapid delayed rectifier current, IKr. In each case, we progressively increased the magnitude of the insult and documented the level that produced arrhythmic dynamics , 4.1 mM, 2.9 mM, and 2.5 mM. Simulation time courses (top) and exemplar AP recordings (bottom) are shown in +] in Cell Line A, consistent with the increased susceptibility predicted by simulations, whereas normal dynamics were observed at all levels of [K+] in Cell Line B, consistent with the predicted protective effect.To test the surprising predictions that particular TKIs could either increase or decrease susceptibility to arrhythmogenic insults, depending on cell line, we recorded APs and intracellular [Caizations . Experim+]. Consistent with expectations, reductions in extracellular [K+] led to increases in Decay Tau and AP triangulation in all groups. These changes, however, were larger in cells treated with the drugs predicted to increase susceptibility, whereas cells treated with drugs in the protective group showed values close to vehicle-treated control cells  whereas negative ΔQ though L-type Ca2+ current ICaL. may contribute to increased arrhythmia susceptibility. These changes, summarized schematically in The results shown above, while demonstrating differences in how individual drugs influence arrhythmia susceptibility between cell lines, do not fully reveal the underlying mechanisms, a question that can be explored through additional model simulations. To address potential mechanisms, we simulated action potentials and ionic currents in iPSC-CMs treated with drugs that increased susceptibility: trametinib and gefitinib in Cell Line A, and trastuzumab and bevacizumab in Cell Line B. At the level of extracellular [Kll lines . For insin vitro experiments, such as those performed with iPSC-CMs, calls into question their direct clinical utility. To attempt to overcome these limitations, we developed an integrative approach that combines the broad coverage provided by transcriptomic measurements in human iPSC-CMs with the mechanistic insights gained from dynamical modeling and physiological experiments. Our underlying premise is that 48 h drug treatments at clinically-relevant concentrations do not cause overt toxicity, but can produce changes in cell state that prime cells for pathophysiological responses to additional stimuli. The results that we obtained, through experimental tests of simulation predictions, corroborate this “two-hit” hypothesis and support our approach to TKI-induced cardiotoxicity as a strategy for screening and improved understanding.The mechanisms underlying cardiotoxicity in some patients treated with TKIs remain unclear, and the elucidation of these mechanisms is a major research focus in cardio-oncology. Although the observed rates of adverse events are relatively low, a literature review conducted in 2017 found that 26 out of then 30 FDA-approved TKIs list some form of serious cardiotoxic effect in their black box warnings . Severalto, or an arrhythmogenic downregulation of currents such as IK1, thereby providing mechanistic grounding for the differences between cell lines  initiative, which aims to improve the testing of drugs for potential risk of Torsades de Pointes due to ion channel block (iPSC-CMs have generated considerable enthusiasm in recent years as a valuable model system for examining drug-induced pathophysiology . Experimel block . When iPel block  and a meel block  to generel block . During el block  provides2+ handling, and contraction. Although this model allowed for straightforward tests of experimental predictions, it only simulates a small part of myocyte biology. As more information becomes available, additional reactions can be quantitatively considered, which will allow for the development of an integrated, multiscale mathematical model that also simulates other processes potentially involved in drug-induced cardiotoxicity, such as hypertrophy (Besides the issue of iPSC-CM maturity mentioned above, several of the study’s limitations suggest avenues that can be pursued in future research on TKI-induced cardiotoxicity. For instance, the expense of comprehensive mRNA sequencing forced us to test only a single concentration of each TKI, with that concentration determined based on estimated plasma concentrations in patients following standard dosing . Althougertrophy  and apopertrophy . ExaminaFinally, we should note that although the differences that were predicted and observed between the two cell lines are intriguing, a much more comprehensive study will be required to characterize drug responses across a population. The strategy we have pursued should help provide a road map for how broad coverage can be balanced with mechanistic rigor in such an investigation.In summary, we have integrated transcriptomic data with mathematical modeling and physiological assays to elucidate individual-specific responses to TKI-induced EC coupling abnormalities. This study is a step forward for individualized drug prescription that can potentially minimize the probability of drug-induced cardiotoxicity.


P < 0.05. (2) The results of cellular experiments showed that overexpression of VASH2 increased the invasion and migration ability of medulloblast Daoy, while knockdown of VASH2 inhibited the invasion and Overexpression of VASH2 upregulated the expression of Smad2 + 3, Smad4, Mmp2 and the apoptotic indicators Bcl-2 and Caspase3, while knockdown of VASH2 suppressed the expression of Smad2 + 3 and Mmp2, and silenced the expression of Smad4 and the apoptotic indicators Bcl2, Caspase3 expression. Flow cytometric cycle analysis showed that VASH2 overexpression increased the S phase in the Daoy cell cycle, while VASH2 knockdown decreased the S phase in the SHH medulloblastoma cell lines DAOY cell cycle. Bioinformatics analysis showed that there was no statistically significant difference between the expression of VASH2 genes in the GSE30074 and GSE202043 datasets and the prognosis of the patients, but the results of this dataset analysis suggested that we need to continue to expand the sample size of the study in the future. The results of the GO enrichment analysis showed that the angiogenic pathway was the most significantly enriched, and the PPI interactions network of VASH2 was obtained from the STRING database. Using the STRING database, we obtained the PPI interaction network of VASH2, and the KEGG enrichment analysis of VASH2-related genes showed that VASH2-related genes were related to the apoptosis pathway, and therefore it was inferred that VASH2 also affects the development of tumors through apoptosis. We found for the first time that the positive expression rate of VASH2 was closely associated with SHH-type pediatric medulloblastoma and that VASH2 was involved in the invasion, migration, cell cycle, and apoptotic capacity of SHH medulloblastoma cell lines DAOY by affecting downstream indicators of the TGF-β pathway. This suggests that it is involved in the progression of pediatric medulloblastoma, and VASH2 is expected to be a diagnostic and therapeutic target for SHH-type pediatric medulloblastoma.To study the differences in VASH2 expression in pediatric medulloblastoma (MB) tumor tissues of different molecular subtypes, to analyze the correlation between VASH2 and the molecular subtypes of medulloblastoma, clinicopathological data, and prognosis, and to explore the specific mechanism of VASH2’s role in SHH medulloblastoma cell lines DAOY. We analyzed 47 pediatric medulloblastoma cases admitted to the Department of Pediatric Neurosurgery of the First Affiliated Hospital of Xinjiang Medical University from January 2011 to December 2019, and the expression levels of YAP1 and GAB1 in these tumor tissues were detected by immunohistochemistry (IHC) and molecularly typed . The correlation between VASH2 and molecular typing of medulloblastoma was analyzed. We also analyzed the medulloblastoma dataset in the GEO database (GSE30074 and GSE202043) to explore the correlation between VASH2 and the prognosis of medulloblastoma patients, as well as performed a comprehensive GO enrichment analysis specifically for the VASH2 gene to reveal the underlying biological pathways of its complex molecular profile. We used vasopressin 2 (VASH2) as a research target and overexpressed and knocked down VASH2 in SHH medulloblastoma cell lines DAOY by lentiviral vectors in vitro, respectively, to investigate its role in SHH medulloblastoma cell lines DAOY cell proliferation, apoptosis, migration, invasion and biological roles in the cell cycle. (1) Among 47 pediatric medulloblastoma cases, 8 were WNT type, 29 were SHH type, and 10 were non-WNT/SHH type. the positive rate of VASH2 was highest in the SHH type with a 68.97% positive rate, followed by non-WNT/SHH and lowest in the WNT type. The results of the multifactorial analysis showed that positive expression of VASH2 was associated with medulloblastoma molecular subtype (SHH type), site of tumor development (four ventricles), and gender ,  Long-term treatment of medulloblastoma remains a great challenge, and clinical research is currently focused on targeted therapy and immunotherapy. These novel therapies have also been gradually applied in clinical treatment, but the prognosis of children with medulloblastoma is still poor compared with the therapeutic effects of other solid tumors, and there is still a long way to go to improve the survival rate and quality of life of children with medulloblastoma2. This suggests that the mechanisms underlying the development of aggressive recurrence in pediatric MB are more complex, and pediatric MB patients still lack objective prognostic assessment indicators and effective therapeutic targets. Therefore, the study of its recurrence mechanism is necessary, and the exploration of its mechanism may provide ideas for the development of new therapeutic targets.Medulloblastoma (MB) is one of the most common and malignant tumors of the central nervous system in children1. Current clinical guidelines for individualized treatment of MB based on these four molecular typologies allow us to further improve the diagnosis of MB in children. After the diagnosis of most pediatric MB, clinicians usually adopt a combination of surgical resection and selective radiotherapy based on postoperative pathologic findings, but the overall efficacy is still unsatisfactory.In 2021, WHO  classified medulloblastoma into four molecular types: WNT-type, SHH-TP53 wild-type, SHH-TP53 mutant, and non-WNT/SHH-type. Among them, the previous group 3 and group 4 types were also categorized as non-WNT/SHH types3. Importantly, the main reason for the poor outcome of tumor treatment is the abnormal formation of tumor blood vessels4.The progression mechanism of childhood MB is the result of a combination of factors, and aggressive tumor recurrence is usually the result of abnormal activation of certain signaling pathways during disease progression. Therefore, it is of great significance to study the genes involved in the progression of childhood MB recurrence and elucidate their mechanisms of action, to find specific target drugs and prognostic molecular markers for the clinical treatment of childhood MB. Our analysis of the latest findings revealed that tumor recurrence is related to the biological processes of tumor cell proliferation, invasion, and apoptosis, and is also closely related to the treatment and prognostic assessment of the disease5.Antiangiogenic therapy has emerged as a potential strategy for the treatment of pediatric medulloblastoma. Angiogenesis is one of the hallmarks of tumor progression. Angiogenesis is a dynamic process and neovascularization is usually regulated by both stimulatory and inhibitory factors. However, in the pathological state of a tumor, this “angiogenic balance” may become imbalanced; dysregulation or overproduction of angiogenesis-inducing factors often leads to excessive formation of abnormal blood vessels. Recent studies have found that endogenous angiogenesis inhibitors produced by endothelial cells can regulate angiogenesis through both positive and negative feedback, and VASH2 (Vasohibin-2) belongs to this group of regulators6. Researchers have found that VASH2 promotes the progression of solid tumors such as pancreatic and ovarian cancers through the process of epithelial–mesenchymal transition 7. Gene expression analyses have shown that VASH2 is expressed in endothelial cells during human and mouse embryonic development, and these expressions disappear after birth8. VASH2 is expressed in monocytes, and monocyte infiltration occurs at the anterior end of neovascular sprouting and promotes angiogenesis. It has been shown that VASH2 is abnormally expressed in solid tumor cells such as gastric cancer, hepatocellular carcinoma, and ovarian plasmacytoid malignancies9 and that VASH2 plays an important role in the process of carcinogenesis.The family of angiogenesis inhibitors includes VASH1 (Vasohibin-1) and VASH2, where VASH1 is a negative regulator of angiogenesis and inhibits angiogenesis; VASH2, a homolog of VASH1, acts in the opposite direction of VASH1 and promotes angiogenesis during repair of damage in the bodyIt is well known that childhood medulloblastoma is a tumor of embryonic origin, and medulloblastoma originates from embryonic stem cells that differentiate into neural and supportive cells. However, in some cases, the normal differentiation process of these cells is interfered with, causing them to proliferate abnormally and form tumors. The same relationship exists between the VASH2 gene and the embryo. VASH2 is a protein encoded by the human gene VASH2. It plays an important role in embryonic development and angiogenesis. Studies have shown that the VASH2 protein plays a key regulatory role in the early stages of embryonic development. It is involved in embryonic angiogenesis and the development of the vascular system. Specifically, the VASH2 protein is expressed in vascular endothelial cells and is involved in the regulation of angiogenesis and vascular density. It regulates the process of angiogenesis by inhibiting the action of vascular endothelial growth factors, such as VEGF.In addition, the VASH2 protein has been implicated in the development of the nervous system during embryonic development. It has been found that the expression of the VASH2 protein plays an important role in regulating the nerve growth cone and neuronal migration in the embryo. It can affect neuronal growth and differentiation and is involved in the formation and connectivity of the nervous system. In other studies, it has been shown that VASH2 promotes tumor growth to a certain extent during tumor progression, but since tumor progression is a complex process, VASH2 it' s promoting role deserves to be studied in depth, based on the above research background we chose VASH2 and medulloblastoma as a research object.In this study, we collected paraffin specimens of 47 pediatric medulloblastoma tissues treated in the Department of Pediatric Neurosurgery of the First Affiliated Hospital of Xinjiang Medical University from January 2011 to December 2019, approved by the ethics committee of the First Affiliated Hospital of Xinjiang Medical University , and all children were not treated with radiotherapy, chemotherapy, or immunotherapy before surgery, with a postoperative follow-up of 40 The follow-up period after surgery was 40–65 months. The study complied with the 2013 revised Declaration of Helsinki and written informed consent was obtained from all patients.SHH medulloblastoma cell lines DAOY were purchased from the cell bank of the Chinese Academy of Sciences and preserved and cultured in the cell laboratory of the Clinical Research Institute of Xinjiang Medical University.VASH2 antibody (PA5-113065) was purchased from Thermo Fisher, USA. GAB1 antibody (ab59362), YAP1 antibody (ab114862), Smad4 antibody (ab230815), Smad2 + 3 antibody (ab207447), Bcl-2 antibody (ab117115), Caspase3 antibody (ab2302), β-actin antibody (ab8227), purchased from Abcam Inc, USA. Fetal bovine serum FBS, RPMI1640 basal medium, trypsin, and mycin-streptomycin double antibodies Gibco, USA, RPMI1640 medium purchased from Gibco, USA. AEC enzyme substrate kit and crystalline violet powder were purchased from Zhongshan JinQiao, China. Cell counting kit 8 purchased from Bioss Beijing, China. Transwell chambers and substrate gel (356234) were purchased from Corning, USA. MMP-2 antibody (bs-4605R) was purchased from Bosun, China. VASH2 overexpression lentivirus (42448-2), negative control virus Con238, and VASH2 knockdown lentivirus (1023B4-1), Target Seq: GCTCCAGGCGATCCAGAATTA. Negative control virus LVCON313 was purchased from Genechem, China. Purimycin solution (A610593) and BSA (A602440-0050) were purchased from Shanghai Biotech, China.2 cell culture incubator, and the cells were passaged when the cell fusion reached about 85%, and SHH medulloblastoma cell-lines DAOY were transfected with lentiviral vectors provided by Genechem. medulloblastoma cell lines DAOY.SHH medulloblastoma cell-lines DAOY were cultured in RPMI 1640 medium containing 10% fetal bovine serum at 37 °C in a 5% COThe cells were divided into a control group: NC group, VASH2 overexpression: VASH2-OE group, and VASH2 knockdown group: VASH2-si group. . After 48 h of lentiviral transfection, the lentiviral transfection effect was observed using fluorescence microscopy. After confirming the cell transfection efficiency, the medium was changed to a complete medium. After that, the transfected cells were screened for the stable strain with 2 μg/mL puromycin. Subsequently, control cell lines with stable growth, VASH2 overexpressing medulloblast cell lines, and VASH2 knockdown medulloblast cell lines were obtained.All paraffin sections were preheated for 0.6 h at 56 °C, dewaxed in xylene, and rehydrated by graded alcohol. Antigens were recovered by heating in citrate buffer (pH 6.0) for 20 min and quenched in a methanol and hydrogen peroxide bath for 30 min to quench endogenous peroxidase activity. Samples were then incubated overnight at 4 °C with mouse antibodies and biotinylated goat anti-mouse or rabbit IgG antibodies labeled with streptavidin-horseradish peroxidase (HRP) using a AEC staining kit according to the manufacturer's instructions. Five representative high-magnification fields (400 × magnification) are selected for each tissue section for histological evaluation. For each protein, two parameters, positive rate (PR) and staining intensity (SI) were used to describe the range and intensity of expression based on the positively stained cells in the sample. The scoring criteria were: positive staining for GAB1 and YAP1, and VASH2 showed only cytoplasmic staining with dark red granules. According to the specific detailed antibody instructions, positive and negative control, experimental sections were set up. Each pathological section was randomly selected by two pathologists in 10 fields of view under high magnification, and the results were judged by two scoring indexes, including the percentage of positive cells and staining intensity, in addition to the determination of target protein localization. Staining intensity was scored: 0 for no staining, 1 for light red, and 2 for dark red; the percentage of positively expressing cells was scored: 0 for ≤ 5%, 1 for 6–25%, 2 for 26–50%, and 3 for more than 50%. The 2 scores were multiplied and 0–2 was considered negative and greater than or equal to 3 was considered positive.Cells were harvested and washed twice with PBS , and total protein was extracted with RIPA buffer . Approximately 30 µg of total protein was subjected to SDS-PAGE and transferred to PVDF membranes, which were then blocked with 5% skim milk in TBST and incubated with primary antibody (1:1000) containing 5% BSA overnight at 4 °C. The membranes were washed three times with TBST and incubated with HRP-coupled secondary antibody  for 2 h at room temperature using enhanced chemiluminescence reagents  exposure and then re-probed-with-anti-β-actin-antibody  at a dilution of 1:2000 to confirm the same protein loading. Detection of VASH2, Smad4, Smad2 + 3, Mmp2, Bcl-2, Caspase3.3) were inoculated into 96-well plates and incubated with CCK-8 solution  at different periods  and 37 °C for 2 h. Cell viability was expressed by absorbance measured at 450 nm.The transfected groups of cells  with an 8-μm pore size polycarbonate filter membrane. The lower chamber contains a medium containing 10% FBS. Cells were incubated for 48 h. Cells on the lower surface of the membrane were then fixed in methanol and stained with 1% crystal violet. The stained membranes were photographed by microscopy and the invading cells were counted. The experiment was repeated at least 3 times.Matrigel and Transwell were used to perform SHH medulloblastoma cell lines DAOY invasion assays to construct invasion chambers for the separation of highly invasive and less invasive cells. Cells were inoculated in Matrigel at a density of 1 × 105 cells were resuspended in 100 µL PBS before washing and fixation with 4% paraformaldehyde. no cells were permeabilized because all protein markers were membrane proteins. Samples were analyzed by BD FACS Aria flow cytometry (BD Biosciences).The concentration of antibody used for staining and the co-incubation time were calculated according to the manufacturer’s instructions. 12.5 µg/mL of PE anti-human flatfoot protein antibody was used. 1 × 10https://www.r-project.org) was used for statistical analysis and mapping. The STRING database was utilized to obtain the PPI interactions network of VASH2, and the VASH2-associated genes were enriched and analyzed.Obtain the GSE30074 and GSE202043 datasets (medulloblastoma samples) and their corresponding clinical information from the GEO website. Precisely aligned gene expression matrices and added annotations to genes from the platform annotation files to reveal the expression levels of the VASH2 gene in the medulloblastoma domain. Rigorously collect clinical data, exclude samples lacking survival data, and harmoniously integrate survival statistics with VASH2 gene expression profiles. Patients were expertly divided into two distinct cohorts based on the median expression level of the enigmatic VASH2 gene and analyzed in depth by the highly respected Kaplan–Meier method, presenting the data in a beautiful KM curve. At the same time, a comprehensive GO enrichment analysis was performed specifically for the VASH2 gene, revealing the underlying biological pathways of its intricate molecular pedigree. R software  method was used to compare differences between the two groups. Statistical significance was set at P < 0.05.Data were analyzed using SPSS 17.0. Quantitative data were expressed as mean ± standard deviation, and Student's This study was approved by the Ethics Committee of Xinjiang Medical University , and written informed consent was obtained from the patients.Molecular typing of medulloblastoma tissues was performed according to immunohistochemistry, and the results were based on the positivity of GAB1 and YAP1 as the typing, with both GAB1 and YAP1 positive as SHH type, GAB1 negative and YAP1 positive as WNT type, and both GAB1 and YAP1 negative as non-WNT/SHH type. Of the final 47 patients, 8 were WNT, 29 were SHH, and 10 were non-WNT/SHH. See Fig. VASH2 was expressed to varying degrees in the tissues of all three subtypes of childhood medulloblastoma. 25 cases of VASH2 were positive and 22 cases were negative in 47 cases of childhood medulloblastoma, a positive rate of 53.19%; VASH2 was positive in 2 cases, and negative in 6 cases of 8 cases of WNT-type medulloblastoma, a positive rate of 25%. 20 cases of VASH2 were positive and negative in 29 cases of SHH-type medulloblastoma, with a positive rate of 68.97%, and VASH2 was positive in 3 cases and negative in 7 cases of 10 cases of non-SHH/WNT-type medulloblastoma, a positive rate of 30%. VASH2 was positive in 20 cases of SHH-type medulloblastoma and negative in 9 cases, with a positive rate of 68.97%, and VASH2 was positive in 3 cases of non-SHH/WNT-type medulloblastoma and negative in 7 cases, with a positive rate of 30%. lowest. See Table P > 0.05) concerning patient non-WNT/SHH type, age, whether the tumor metastasized, survival months, survival outcome, whether radiotherapy was given, and whether chemotherapy was given; the difference in VASH2 expression in pediatric medulloblastoma tissue was statistically significant (P < 0.05) concerning patient SHH type, fourth ventricle, and gender . tumor tissues showed statistically significant differences in expression with patient SHH type, fourth ventricle, and gender  (P < 0.05). See Table The difference in VASH2 expression in pediatric medulloblastoma tumor tissue was not statistically significant , and the results showed that VASH2 may be a key factor affecting apoptosis in SHH medulloblastoma cell-lines DAOY. This also supports the results of the immunohistochemistry experiments in terms of molecular mechanisms. See Fig. We used western blot to detect the expression of VASH2, TGFβ pathway downstream indicators Smad2 + 3, Smad4, Mmp2, and apoptosis indicators Bcl2 and Caspase3 protein levels in SHH medulloblastoma cell lines DAOY after overexpression and knockdown of VASH2, respectively, using β-actin as an internal reference. The results showed that Smad2 + 3, Smad4, Bcl2, and Caspase3 expression was changed in SHH medulloblastoma cell lines DAOY regardless of overexpression or knockdown of VASH2, indicating that VASH2 expression in myeloblasts may be mediated by affecting downstream indicators of TGFβ pathway. The expression of Bcl2 and Caspase3 in the VASH2-OE group was the most significant difference compared to the other two groups (P < 0.05), suggesting that overexpression of VASH2-OE significantly promoted the migration and invasion ability of SHH medulloblastoma cell-lines DAOY, and VASH2-si-RNA significantly inhibited the migration and invasion ability of medulloblast migration and invasion ability of SHH medulloblastoma cell-lines DAOY, (P < 0.05). See Fig. We used Transwell chambers to detect the effects of VASH2 overexpression and VASH2 silencing on the migration and invasion ability of SHH medulloblastoma cell lines DAOY. By counting the cells in the VASH2-OE group versus the VASH2-si-RNA group and the cells in the empty NC group that penetrated the basement membrane of the Transwell. The results showed that the number of cells penetrating the basement membrane of Transwell was higher in the VASH2-OE group than in the VASH2-si-RNA and empty-loaded groups, and the difference was statistically significant (P > 0.05) thus it is clear that modulation of VASH2 does not affect the proliferation ability of SHH medulloblastoma cell lines DAOY. See Fig. The proliferation ability of cells in the VASH2-NC, VASH2-OE, and VASH2-si groups was examined using the CCK-8 assay (Cell Counting Kit-8). The results of the assay showed no statistical difference in the proliferation ability of the three groups, P > 0.0 thus it P < 0.05; meanwhile, the proportion of S phase of cells in the VASH2-OE group (51.57 ± 0.74%) was higher than that in the VASH2-si group (20.54 ± 1.27%) and the VASH2-NC group (35.85 ± 1.34%), and the differences were statistically significant (P < 0.05), i.e., relative to the VASH2-si and VASH2-NC groups, the cell cycle of the VASH2-OE group was blocked in S phase, with prolonged DNA synthesis and active cell division. The results suggest that overexpression of VASH2 in SHH medulloblastoma cell lines DAOY promotes medulloblast proliferation, and VASH2 knockdown inhibited the proliferation of myeloblasts. See Fig. Flow cytometry was used to analyze the alteration of the SHH medulloblastoma cell lines DAOY cell cycle by overexpression and silencing of the VASH2 gene. Flow cycle results showed that overexpression of VASH2 decreased the G1 phase of medulloblast cells, while knockdown of VASH2 significantly increased the G1 phase of SHH medulloblastoma cell lines DAOY, 11. The results of the GO enrichment analysis showed that the angiogenic pathway was the most significant, which also supports the conclusion reached in other studies of VASH2. Using the STRING database to obtain the PPI interaction network of VASH2, KEGG enrichment analysis of VASH2-related genes showed that VASH2-related genes were related to the apoptosis pathway, and therefore it was inferred that VASH2 also affects the process of tumorigenesis and development through apoptosis12. See Figs. Bioinformatics analysis showed that K-M survival analysis of VASH2 gene expression and patient prognosis in the GSE30074 and GSE202043 datasets still did not show any statistical difference, but the results of this dataset analysis suggest that we need to continue to expand the sample size of the study in the future13. Even after surgical resection, radiotherapy and chemotherapy may cause a series of complications such as impaired cognitive function, which seriously affect the long-term prognosis of children and bring a heavy burden to their families and society. With the advancement of science and technology, the concept of precision and individualized treatment of tumors has been proposed, and the exploration of efficient and safe diagnostic and therapeutic target development in pediatric neuro-oncology is particularly important14. Since humans entered the molecular era, the application of deep sequencing technology in tumor research has enabled researchers to have a clearer understanding of tumors and more concepts of tumor molecular subtypes have been proposed, providing theoretical support for precision therapy15. WHO redefined pediatric medulloblastoma in 2021 into four subtypes: WNT, SHH-TP53 wild type, SHH-TP53 mutant type, and non-WNT/SHH type, incorporating the previous group3 and group4 types into the non-WNT/SHH type1. SHH-type medulloblastoma has been reported to have the highest incidence among patients with all types of medulloblastoma, about 1/3, and is prone to early metastasis. SHH-type medulloblastoma is often accompanied by abnormal activation of the SHH signaling pathway, and studies have shown that abnormal activation of this pathway often affects normal cerebellar development16. In other solid tumors, abnormal activation of the SHH pathway leads to tumorigenesis, such as cutaneous basal carcinoma and digestive tract tumors17. Also, abnormal expression of proteins downstream of this pathway  is importantly associated with the development of SHH-type medulloblastoma. The prognosis of children with SHH-type medulloblastoma varies from individual to individual, with patients with SHH-TP53 mutation having the worst prognosis, while patients with SHH-TP53 wild type have a relatively better prognosis18. Therefore, it is important to implement individualized and precise treatment for patients with SHH medulloblastoma and explore new targets for diagnosis and treatment. As reported earlier, the treatment of SHH-type medulloblastoma was mainly developed for inhibitors of SHH-related signaling pathway proteins, and satisfactory efficacy was achieved in previous clinical trials19. In the present study, children with SHH type likewise accounted for the highest proportion of cases. Currently, there are no reports on the role and mechanism of VASH2 in CNS tumors, especially in pediatric medulloblastoma, both at home and abroad; therefore, it is important to investigate the correlation between VASH2 and the pathogenesis of SHH-type medulloblastoma to enhance the prognosis of children with SHH-type medulloblastoma.Childhood medulloblastoma is characterized by high aggressiveness, easy recurrence, and poor prognosisIn this study, we examined the expression level of VASH2 in pediatric medulloblastoma tissues by immunohistochemistry and found that the positive expression rate of VASH2 was closely related to the molecular typing of medulloblastoma. Subsequently, we performed VASH2 regulatory modeling experiments in SHH medulloblastoma cell lines DAOY with two medulloblastoma datasets GSE30074 and GSE202043 (medulloblastoma samples), and their corresponding clinical information obtained from the GEO database. Kaplan–Meier analysis was performed to present the data as Kaplan–Meier curves. At the same time, a comprehensive GO enrichment analysis was performed specifically for the VASH2 gene, revealing the underlying biological pathways of its intricate molecular pedigree. Finally, the STRING database was utilized to obtain the PPI interactions network of VASH2, and the enrichment analysis of VASH2-associated genes was performed. The findings on the dataset partially support our conclusions and also suggest that a more comprehensive revelation of the specific mechanism of action of VASH2 in pediatric medulloblastoma in the future will require us to not only increase the sample size, but also to carry out studies in vivo, in vitro, and from medulloblastomas with different molecular typing.21. One study showed that the knockdown of VASH2 significantly increased the chemosensitivity of tumor cells to paclitaxel PTX22. VASH2 increased the expression of ZEB1/2 in pancreatic tumors through activation of the SHH pathway, which both facilitated the EMT process and increased the resistance of pancreatic tumors to gemcitabine chemotherapy23. The same conclusion was reached in another study on liver tumors24. In addition, in a study on non-small cell lung cancer, researchers found that VASH2 promoted tumor cell proliferation and resistance to adriamycin through the AKT signaling pathway25. As shown in earlier studies, microtubules, as an important component of the eukaryotic cytoskeleton, are involved in a variety of cellular functions26. VASH2 has an atypical tyrosine-catalyzed structure that affects post-translational modification of microtubule proteins in the cell and is involved in tyrosine recycling and degradation27. However, dysregulation of tyrosine cycling is strongly associated with disease and cancer29. A study reported in Nature showed that microtubule-affinity-regulated kinase 4 (MARK4) regulates the contractility of cardiac myocytes by promoting the phosphorylation of microtubule-associated protein 4 (MAP4), which facilitates entry of vasodilator 2 (VASH2) into the microtubule for α-microtubule protein de-tyrosination29. contractility of cardiomyocytes30. Conversely, inhibition of microtubule deacetylation improves contractility in failing cardiomyocytes, suggesting that VASH2 is also closely related to the pathogenesis of heart failure32.The current study preliminarily elucidated the biological function of VASH2 in SHH medulloblastoma cell-lines DAOY, and conducted a preliminary exploration of its related signaling pathways, providing a theoretical basis for exploring new diagnostic and therapeutic targets for pediatric medulloblastoma. In a recent study report, researchers found that the expression level of VASH2 was abnormally elevated in some solid tumors such as gastric cancer, breast cancer, hepatocellular carcinoma, and pancreatic cancer, and promoted tumor angiogenesis under paracellular secretion, which was closely related to tumor prognosis33. In the G1 phase, cells synthesize RNA and proteins, whereas, in the S phase, cells mainly undergo DNA synthesis35. In the present study, we found that modulation of VASH2 overexpression in SHH medulloblastoma cell lines DAOY increased the S phase in the Daoy cell cycle, whereas knockdown of VASH2 shortened the S phase in the Daoy cell cycle. VASH2 can affect DNA synthesis in the Daoy cell cycle.In this study, we explored the potential correlation between the high expression level of VASH2 and the pathological features of medulloblastoma  using immunohistochemical techniques, and we also found that the lowest expression level of VASH2 was found in children with WNT-type medulloblastoma, suggesting that VASH2 may be a potential diagnostic target for predicting the molecular subtype of medulloblastoma. In addition, the cell cycle consists of interphase and division (M phase), and division consists of three phases: pre-DNA synthesis (G1 phase), DNA synthesis (S phase), and late DNA synthesis (G2 phase)37. Apoptosis plays an important role in many biological developmental processes in organisms, such as the turnover of old and new cells in the body, biological embryonic development, disease, and tumor progression39. In most tumors, the unlimited proliferation of tumor cells is inextricably linked to apoptosis40. The results of this study showed that overexpression of VASH2 in myeloblastoma cells Daoy led to a significant increase in the expression level of Bcl2 (B-cell lymphoma/leukemia-2 gene) in myeloblastoma cells, whereas knockdown of VASH2 significantly inhibited the expression of Bcl2 in SHH medulloblastoma cell-lines DAOY. Meanwhile, the trend of caspase3 in medulloblastoma cell lines DAOY after modulation of VASH2 was consistent with that of Bcl2, a proto-oncogene that inhibits apoptosis, and earlier studies have shown that Bcl2 is a downstream target gene of the SHH signaling pathway, and the expression level of SHH in medulloblastoma cell-lines DAOY was significantly increased by knockdown of VASH2, while the expression level of Bcl2 in SHH medulloblastoma cell-lines DAOY was significantly inhibited by knockdown of VASH2. SHH signaling pathway in DAOY is in turn derived from SHH-type medulloblastoma42. Overexpression of Bcl2 in cells protects against Fas ligand-induced cell death43. The results of basic research have shown that Bcl2 enhances the resistance of most tumor cells to DNA damage44, thus improving tolerance to chemotherapeutic agents, but Bcl2 does not promote DNA self-repair45. p53 is also an oncogene, and mutations in this gene occur in more than half of malignant tumors and are also a molecular indicator of DNA damage, and studies have shown that Bcl2 inhibits p53-mediated apoptosis47. The results of one study showed that VASH2 could inhibit apoptosis in liver tumor cells by down-regulating p53, and earlier results showed that the p53 gene was highly expressed in SHH medulloblastoma cell lines DAOY, thus suggesting that VASH2 could also mediate apoptosis in SHH medulloblastoma cell-lines DAOY through the p53 gene48. Caspase3 is an important executor in the apoptotic process. A study showed that overexpression of VASH2 resulted in an increased abundance of intracellular Caspase324. This finding is also consistent with the results of our current study. The above findings suggest that VASH2 can regulate the apoptotic ability in SHH medulloblastoma cell lines DAOY, and VASH2 may be an important regulator in the biological process of medulloblastoma.Apoptosis is a genetically controlled process of programmed cell death and is an active process under the balance of intracellular environmentOne of the most important biological features of malignant medulloblastoma is its aggressive growth. Medulloblastoma cells tend to invade the surrounding normal brain tissues at an early stage, and it is difficult to distinguish them from normal brain tissues with clear boundaries. In this study, we found for the first time that the aberrant expression of VASH2 in pediatric medulloblastoma is closely related to the molecular subtype of medulloblastoma, and VASH2 regulates the invasion, migration, cell cycle, and apoptosis of medulloblastoma mainly through the TGF-β signaling pathway. This suggests that it is involved in the progression of childhood medulloblastoma and plays an important role in the process of medulloblastoma development and progression, and could be a target for medulloblastoma diagnosis and treatment.49, but this method is expensive and has special requirements for the preservation and transportation of tumor tissues, therefore, in this study, we adopted a cost-effective immunohistochemistry method to molecularly type medulloblastoma, and our experiments were mainly conducted using SHH medulloblastoma cell-lines DAOY for the study, this cell line is currently a common model used internationally to study gene function in medulloblastoma and is widely used in many medulloblastoma-related studies. In addition, we used a variety of experimental methods to ensure the reliability and reproducibility of our findings. Although we initially realized that the use of a single cell line might limit the depth and breadth of our study, and we were unable to obtain other cell lines suitable for our study under the current circumstances, we will try to use other medulloblastoma cell lines for simultaneous validation in our future studies, and we will also carry out experiments at the animal level for the experimental studies to further strengthen our findings.There are some limitations of this study. Due to the large number of lost cases, the final sample size included in this study was 47 cases, and exploring the prognostic relationship between VASH2 and medulloblastoma still needs to be investigated in a large sample cohort in the future. In addition, the current gold standard for molecular typing of medulloblastoma mainly relies on DNA methylation assayWe found for the first time that the positive expression rate of VASH2 was closely associated with SHH-type pediatric medulloblastoma and that VASH2 was involved in the invasive, migratory, cell cycle, and apoptotic abilities of SHH medulloblastoma cell lines DAOY by affecting downstream indicators of the TGF-β pathway.VASH2 is expected to be a diagnostic and therapeutic target.Supplementary Information.


Graft-versus-host disease (GVHD) remains a major cause of morbidity and mortality after allogeneic stem cell transplantation (SCT). Currently, more patients can receive SCT. This is attributed to the use of reduced intensity regimens and the use of different GVHD prophylaxis that breaks the barrier of human leukocyte antigen, allowing an increase in the donor pool. Once an area with relatively few clinical trial options, there has been an increase in interest in GVHD prophylaxis and treatment, which has led to many US Food and Drug Administration (FDA) approvals. Although there is considerable excitement over novel therapies, many patients may not have access to them due to geographical or other resource constraints. In this review article, we summarize the latest evidence on how we can continue to repurpose drugs for GVHD prophylaxis and treatment. Drugs covered by our review include those that have been FDA approved for other uses for at least 15 years (since 2008); thus, they are likely to have generic equivalents available now or in the near future. There has been an increase in the use of PTCy in different donor types after its successful use in haploidentical SCT . In the  in GVHD . PTCy wa in GVHD . In mous in GVHD . In addi in GVHD –19, the  in GVHD  due to d in GVHD –22.After the success in the haploidentical setting, many retrospective studies have examined PTCy use in mismatched unrelated donors (MMUDs) –25. A prth ASH Annual Meeting , platelets ≥ 50,000/mm3 by day +100 , and lymphocytes ≥ 1,000/mm3 by 1 year . In the MRD or MUD PB but with non-myeloablative setting, the HOVON-96 trial  inhibitor groups of medications that have been used over many years to control type 2 diabetes mellitus  and apprr-beta-1 . In addir-beta-1 .Farag et al. were interested in studying DPP4 inhibitors in improving engraftment post-umbilical cord blood SCT. Interestingly, in the course of conducting their studies, they observed that patients were developing GVHD at a lower rate than the historical data. This observation led to a formal study of sitagliptin at a dose of 1,200 mg/day in addition to tacrolimus and sirolimus in patients with MRDs or unrelated donors receiving MAC regimens in a phase II non-randomized clinical trial. Sitagliptin was given for 2 weeks starting the day of the transplant, while tacrolimus and sirolimus were tapered at day 100 and discontinued by approximately day 180. In the event of renal toxic effects, MMF  was substituted for tacrolimus at the physicians’ discretion. By day 100, only two of 36 patients developed aGVHD, with rates of aGVHD II–IV of 5% and III–IV of 3%. Relapse and NRM were 26% and zero at 1 year, respectively. CI of cGVHD at 1 year was 37%, with seven of the 15 patients who developed cGVHD having moderate cGVHD and three with severe cGVHD. One-year GRFS was 46% . During Interestingly, the effect of sitagliptin on gut microbiome was examined by investigators in mouse models  and in aAnother way to increase the half-life of GLPs including GLP2, an enteroendocrine hormone, is the development of the degradation-resistant GLP2 analogue termed teduglutide. This drug was approved by the FDA in 2012 for short bowel syndrome. Norona et al.  showed i3.2AAT is a plasma glycoprotein that is produced by hepatocytes. It has a unique three-dimensional structure  that allMarcondes et al.  showed r3.3Vitamin A is involved in many biological processes including the regulation of immune responses via targeting T cells, B cells, antigen-presenting cells, and innate lymphoid cells, with most of its effect being related to its major metabolite retinoic acid (RA) . RA signth percentile of normal for age were randomized to receive high-dose oral vitamin A or placebo pre-SCT  and then went up to 1,000 units hCG/m2 (level 2) and 2,000 units hCG/m2 (level 3) given subcutaneously every other day for 1 week in the high-risk, newly diagnosed aGVHD and 2 weeks in the second-line aGVHD. Patients with complete or partial response were allowed to continue maintenance dosing of uhCG twice weekly for 5 weeks. Treatment was tolerated except for adverse events expected in these patients with GVHD, and only edema was considered a treatment-related adverse event. In the phase II report , which is different from other usual kinases because it is inhibited rather than activated by a stimulus. It is also involved in Wnt signaling and recently attracted the interest of many investigators in different fields like Alzheimer’s disease and cardiovascular disease .Beurel et al.  showed t4Benjamin Franklin famously advised fire-threatened Philadelphians in 1736 that “an ounce of prevention is worth a pound of cure”. Prevention of GVHD not only helps decrease health care expenditure but also improves the quality of life for SCT recipients. However, prevention of GVHD might not be one-size-fits-all. There are so many variables that should be taken into consideration when thinking of GVHD prevention, especially when more options will be available. For example, in high-risk malignancies in pediatrics with ages 18 and younger, with MAC BM PTCy, there was a higher relapse probability (49%) compared to children historically receiving CSA/MTX  , possiblIn the treatment of GVHD, more knowledge about pathophysiologic mechanisms in immune and regenerative pathways, in addition to the microbiome and enteroendocrine systems of the gut, may help provide patients with innovative strategies in the first-line treatment of GVHD. This is especially important for high-risk patients who have historically received prolonged high-dose corticosteroids. This is a huge unmet medical need. Filling this gap will help decrease immunosuppression, improve responses, and increase quality of life.The process of traditional drug development is a time-consuming and expensive process that includes 2–5 years of preclinical studies and toxicology, followed by a minimum of 3–5 years of phase I–III clinical trials, before entering the registration and marketing phases Figure 1SF and SH designed, wrote, and edited the manuscript. All authors contributed to the article and approved the submitted version.


Ferroptosis suppressor protein 1 and glutathione peroxidase 4 have been identified as key molecules in two independent pathways associated with ferroptosis inhibition. This study investigated the prognostic significance and clinical associations of FSP1 and GPX4 expression in esophageal squamous cell carcinoma (ESCC) and assessed the therapeutic potential of regulating these molecules in ESCC cells.Immunohistochemical analysis was performed on surgical specimens of 97 patients with ESCC for FSP1 and GPX4 expression. To identify the change in ESCC cell viability, FSP1 and GPX4 inhibitors were administered to three cell lines. In addition, ferroptosis as the cause of reduced cell viability by FSP1 and GPX4 inhibition was confirmed.p < 0.001). In multivariate analysis, positivity for both FSP1 and GPX4 was an independent poor prognostic factor (p = 0.002). The combination of FSP1 and GPX4 inhibitors induced cell death more potently than each inhibitor did alone. Furthermore, the ferroptosis inhibitor markedly canceled this cell death.Prognosis was significantly worse for patients in the group positive for both FSP1 and GPX4 compared with the other groups (Overexpression of FSP1 and GPX4 is a poor prognostic factor for patients with ESCC. Simultaneous suppression of both FSP1 and GPX4 caused potent cell death, which was markedly abrogated by ferroptosis inhibitors. These findings indicate that simultaneous regulation of FSP1 and GPX4 may be a new therapeutic target in ESCC.The online version contains supplementary material available at 10.1007/s10388-022-00982-x. Esophageal cancer is the sixth most common cancer globally, with more than 480,000 new cases annually . MoreoveInducing cancer cell death is an important strategy in chemotherapy. Chemotherapeutic drugs for esophageal cancer include 5-fluorouracil (5-FU), platinum, and taxane , all of In 2012, researchers discovered ferroptosis, defined as cell death by iron-dependent lipid peroxidation reaction . ResearcWe previously reported that high expression of GPX4 correlated with worse prognosis in ESCC . The preStudy participants were 97 patients who underwent radical esophagectomy for ESCC at Tottori University Hospital, Yonago, Japan, from January 2009 to December 2017. Stored paraffin-embedded specimens were obtained for immunohistochemical analysis. Patients with multiple primary cancers were excluded. Pathological diagnosis was confirmed according to the Japanese Classification of Esophageal Cancer . The patImmunohistochemistry was performed according to the standard protocols, which are further described in the Online Resource.The expression of FSP1 was defined as < 20% negative and ≥ 20% positive in terms of the tumor-stained area. Immunolabeling was evaluated by three investigators ; consensus was reached in all cases. To evaluate GPX4 expression, our previous staining results were used as follows : “ExpresThree human ESCC cell lines, including KYSE30, KYSE510, and KYSE520, were grown in Roswell Park Memorial Institute Medium supplemented with 10% fetal bovine serum. All cell lines were maintained under 37 °C in atmospheric air supplemented with 5% carbon dioxide and passaged at a ratio of 1:3–1:10 every 2–3 days. Cell line and reagent details are described in the Online Resource. Clinical information on these cell lines is summarized in Table S1 in the Online Resource.5 cells in four 6-cm dishes and incubated overnight. After 24 h, the cells were lysed and protein extracted. Detailed descriptions of the subsequent steps are included in the Online Resource.In this analysis, the KYSE30, KYSE510, and KYSE520 cells were seeded at 2 × 10ESCC cell lines were treated with iFSP1—an FSP1 inhibitor—and -RSL3  alone or in combination. Cell proliferation was assessed using the Cell Counting Kit-8 (CCK8) according to the manufacturer’s protocol, which is further detailed in the Online Resource.The procedure and elapsed time from cell seeding, addition of drugs, and cell survival evaluation are the same as the cell proliferation assay. The added drug included a combination of iFSP1 and RSL3 plus liproxstatin-1  or Z-VAD-FMK Caspase Inhibitor VI  or Necrostatin-1 , respectively. Detailed descriptions of the subsequent steps are included in the Online Resource.p values < 0.05 were considered statistically significant. Statistical analyses were performed using GraphPad Prism  and SPSS version 25.0 .For overall survival and relapse-free survival, survival curves were estimated by Kaplan–Meier analysis; differences in survival curves were compared using the log-rank test. Cox proportional hazards models were used for univariate and multivariate analyses. All quantitative values are presented as the median. For FSP1 and GPX4, association between expression and patient's characteristics was examined using the chi-squared test and Fisher exact test for categorical variables. All Expression of FSP1 and GPX4 in resected specimens from patients with ESCC was evaluated by immunohistochemistry and classified as positive or negative based on the percentage of positively tumor-stained area  and disease stage (p = 0.029) than the FSP1-negative group. The GPX4-positive group had significantly higher invasion depth (p = 0.014), lymph node metastasis (p = 0.001), lymphatic involvement (p = 0.019), vascular involvement (p = 0.011), and disease stage (p = 0.002) than the GPX4-negative group  were positive for FSP1 and 40 (41.2%) for GPX4. The FSP1-positive group had a significantly higher invasion depth   . The prognostic comparison of the patients with ≥ T2 or lymph node metastasis among the same three groups categorized according to the FSP1 and GPX4 expression revealed a pattern that was similar to that observed in the entire patient population . In multivariate analysis, positivity for both FSP1 and GPX4 was an independent poor prognostic factor Supplementary file 2 (TIF 590 KB)Below is the link to the electronic supplementary material.