Corticosteroids are the first line therapy for acute graft-versus-host disease (GVHD). However, the outcome of steroid refractory GVHD (SR-GVHD) is poor due to a lack of effective treatments. The development of therapies for SR-GVHD is limited by an incomplete understanding of its pathophysiology partly because of the absence of clinically relevant animal models of SR-GVHD. Here we addressed the need for a SR-GVHD animal model by developing both MHC matched multiple minor histocompatibility antigens (miHAs) mismatched and MHC mismatched haploidentical murine models of SR-GVHD. We demonstrate that animals can develop SR-GVHD regardless of whether steroids are initiated early or late post allogeneic bone marrow transplantation . In general, we observed increased GVHD specific histopathological damage of target organs in SR-GVHD animals relative to steroid responsive animals. Interestingly, we found no significant differences in donor T cell characteristics between steroid refractory and responsive animals suggesting that donor T cell independent mechanisms may play more prominent roles in the pathogenesis of SR-GVHD than was considered previously. Many agents2 have shown encouraging phase II response rates, but none have demonstrated a long term survival advantage including three tested in randomized controlled trials3. The lack of effective SR-GVHD treatments is likely compounded by our incomplete understanding of its pathophysiology. There are many reasons why SR-GVHD pathogenesis is poorly understood, but one reason may be the lack of clinically relevant animal models. Herein we generated murine SR-GVHD models, which surprisingly suggest that donor T cell independent mechanisms may play a more important role in SR-GVHD pathogenesis than previously appreciated.Acute graft-versus-host disease (GVHD) is a major complication of allogeneic cell transplantation . For decades, steroids have remained the first-line treatment for acute GVHD; however, 30–50% of patients develop steroid refractory GVHD (SR-GVHD), which portends an extremely poor prognosis5 so as to better reflect the prednisolone-equivalent dose used clinically. Surprisingly, despite dexamethasone treatment, there were no significant differences in overall survival (OS), GVHD clinical scores, or the percentages of body weight (BW) lost between treated and untreated control groups in our model  matched, we first utilized the major histocompatibility (MHC) matched multiple minor histocompatibility antigens (miHAs) mismatched C3H.sw→B6 BMT model. Recipient B6 WT animals were lethally irradiated and transplanted with splenic T cells along with bone marrow (BM) cells from either syngeneic B6 or allogeneic C3H.sw donors. Dexamethasone (DEX) was delivered from day +7 to day +21 at a dose of 0.1 mg/kg Fig.  which wadel Fig. . To deteBecause we couldn’t see any therapeutic responses in DEX treated animals, we speculated that treated animals have different responses to DEX in spite of a uniform genetic background and housing conditions. Interestingly, we observed three phenotypes in response to DEX: 1) progressive GVHD characterized by severe peak GVHD clinical scores, progressive BW loss and early mortality; 2) stable GVHD characterized by moderate GVHD clinical scores, moderate BW loss, and minimal late mortality; and 3) steroid-responsive GVHD characterized by near complete normalization of GVHD clinical scores, near complete normalization of BW loss, and no mortality Fig. . We wereTo eliminate strain dependent factors, we also examined whether the differential steroid response predicted by our scoring systems holds in an MHC mismatched haploidentical B6→B6D2F1 model. We found a similar differential response to corticosteroids; albeit with a slightly different distribution among the groups and greater mortality in the St and Res groups Fig. .To test whether our scoring system specifically separates DEX treated animals relative to control untreated animals, we applied our scoring system to 15 untreated control animals shown in Fig. To account for any confounding effect of radiation toxicity, we next sought to determine whether steroid refractoriness can be modelled at a later time-point. To accomplish this, we utilized the same C3H.sw → B6 model except that DEX was administered from day +21, at which point there was already approximately 20% mortality among allogeneic recipients, to day +35 Fig. . We agai7, we determined whether these cytokines correlated with treatment responses. To our surprise, serum levels of IFN-γ, and IL-17A were not significantly altered in any early or late DEX-treated animals , proinflammatory cytokine production , and CD4+CD25+Foxp3+ regulatory T cells (Tregs) by FACS. However, donor CD4+ and CD8+ T cell expansion in the spleen was similar among these groups at day 21 after allo-BMT , memory (CD44+CD62L+), naïve (CD44−CD62L+) T cells, or Tregs in the spleen, but these subsets were similar among all allogeneic groups . Collectively, these data suggest that our SR-GVHD models effectively predict survival and therapeutic responses. Unexpectedly, our data also suggest that SR-GVHD may be mediated by a donor T cell independent mechanism.In this study, we establish steroid refractory murine BMT models using two different clinically relevant BMT models, and two different treatment intervention strategies, early and late DEX treatment. In addition, we used corticosteroid doses that correspond to clinically relevant doses. All of the experiments presented here are required for establishing appropriate models to eventually decipher SR-GVHD pathogenesis and to develop SR-GVHD treatments.9 in Table 10. Therefore, our GVHD scoring system may not be specific nor correlate with histological GVHD grade but this system is used worldwide and consistently reflects murine GVHD severity. Recently, Naserian, et al. proposed a new simple, reproducible, and efficient clinical grading system for murine GVHD relying on the binary evaluation of five visual parameters, including diarrhea, and demonstrated correlation with histological grade11. Future work will seek to assess if Nasarian’s criteria improve on our model of SR-GVHD.Certainly, our models, like any model system have several limitations including the need to be continually optimized to reflect clinical BMT as clinical practice evolves. Nevertheless, we summarize the main differences between our criteria for murine SR-GVHD and clinical SR-GVHD13 following steroid treatment. T cell exhaustion could certainly help explain the clinical differences among our groups. However, why there would be differential exhaustion following steroid treatment in genetically and environmentally homogenous animals is not readily apparent. Nevertheless, our data suggest that immunoregulatory aspects in the context of steroid refractoriness need to be further explored. In addition, these data also suggest that T cell independent mechanisms of tissue tolerance may contribute to SR-GVHD and should be further explored15.To our surprise, we found that donor T cell characteristics were not significant among steroid responsive, refractory, or stable groups. Future studies will seek to elucidate the critical cellular and molecular mechanisms of glucocorticoid response and resistance. One potential explanation for different responses to DEX is differences in pharmacokinetics between groups, which we will explore in future studies. It would also helpful to assess whether donor derived T cells from newly generated bone marrow or those from peripheral expansion are more affected by glucocorticoid treatment. Another consideration to assess is whether donor T cells exhaustion is different among our groups as there are conflicting reports in this regardIn conclusion, we established clinically relevant steroid refractory GVHD murine models. Although we have not shown any critical mechanisms, developing a model is the key first steps. We propose using this model to better understand the pathophysiology of SR-GVHD and to develop new therapeutic and prophylactic GVHD strategies.b, CD45.2+), B6 Ly5.2 , B6D2F1 (H-2b/d), and C3H.sw (H-2b) mice were purchased from the Jackson Laboratory . All animals were cared for according to regulations reviewed and approved by the University Committee on Use and Care of Animals of the University of Michigan, based on University Laboratory Animal Medicine guidelines.C57BL/6  utilizing CD90.2 microbeads (Miltenyi Biotec), and bone marrow (BM) was harvested from donor femurs and tibias. B6 and B6D2F1 animals were used as recipients and received 10–11 Gy (137Cs source) on day −1 and 3 × 106 CD90.2+ T cells along with 5 × 106 BM cells from either syngeneic (B6 or B6D2F1) or allogeneic (C3H.sw or B6) donors on day 0.BMTs were performed as previously described using well established models19. Histopathological analysis of the liver, gastrointestinal (GI) tract, lung, and skin, which are the primary GVHD target organs, was performed as described previously20 utilizing a semiquantitative scoring system implemented by a single pathologist (Chen Liu)20. After scoring, the codes were broken, and the data compiled.We monitored survival after allo-HCT daily and assessed the degree of clinical GVHD weekly, as described previously22. Briefly, to analyze donor T cell or myeloid cell expansion and activation markers, splenocytes from transplanted animals were resuspended in FACS wash buffer  in phosphate buffered saline (PBS)) and stained with conjugated monoclonal antibodies (moAbs). The following antibodies were used: FITC-conjugated moAbs to mouse CD45.1 (clone SF1–1.1) and CD229.1 (clone 30C7); PerCP-Cy5.5-conjugated moAbs to mouse CD4 (clone GK1.5), CD8 (clone 53–6.7) and CD11c (clone N418); PE-conjugated moAbs to mouse CD69 (clone H1.2F3), CD62L (clone MEL-14), and CD11b (clone M1/70); and APC-conjugated moAbs to mouse CD44 (clone IM7) and CD25 (clone PC61). All antibodies were purchased from Biolegend  except the FITC anti-CD229.1, which was purchased from BD bioscience . After staining, the cells were washed twice and fixed with 2% formaldehyde as described previously21. Cells were analyzed using a BD Accuri C6 flow cytometer (BD Bioscience). For intracellular cytokine staining, cells were permeabilized after fixation and stained with PE-conjugated IFN-γ  and granzyme B , APC-conjugated IL-17A  and perforin  according to manufacturer’s protocol.FACS analyses were performed as previously describedTM  or ELISA MAXTM . Assays were performed according to each manufacturer’s protocol and read at 450 nm using a microplate reader . All samples and standards were run in duplicate.Concentrations of IFN-γ and IL-17A were measured in the serum by ELISA with specific anti-mouse MoAbs for capture and detection utilizing BD OptEIAin vitro data, and the Wilcoxon rank test for survival data. A p value <0.05 was considered statistically significant.Statistical significance was determined using the Mann-Whitney U test for Supplementary information


Despite high cure rates, about 20% of patients with advanced germ cell tumors (GCTs) fail cisplatin-based chemotherapy. High levels of DNA methylation have been identified in GCTs and linked to cisplatin resistance. Here, we examined the effects of DNA hypomethylating 5-azacitidine (5-aza) on two embryonal carcinoma cell lines  and their cisplatin-resistant isogenic derivatives. Effects on cell viability and cisplatin sensitivity were assessed by the trypan blue exclusion method. Western blotting was used to examine induction of apoptosis 5-aza and results were validated by flow cytometry. Single agent treatment with 5-aza strongly impacted viability and induced apoptosis at low nanomolar concentrations, both in cisplatin-sensitive and -resistant cell lines. 5-aza exerted an immediate apoptotic response, followed by a prolonged inhibitory effect on cell viability and cell-cycle progression. Sequential treatment with 5-aza and cisplatin reduced cellular survival of the cisplatin-resistant sublines already at nanomolar concentrations, suggesting a partial restoration of cisplatin sensitivity by the compound. 5-aza demonstrated anti-tumor activity as a single agent at low nanomolar concentrations in GCT cells, irrespective of cisplatin-sensitivity. 5-aza may also have the potential at least to partially restore cisplatin-sensitivity in non-seminoma cells, supporting the hypothesis that combining DNA demethylating agents with cisplatin-based chemotherapy may be a valid therapeutic approach in patients with refractory GCTs. Germ cell tumors (GCTs) are the most common solid malignancy among men up to the age of 45. Histologically, GCTs are classified into undifferentiated, primordial germ cell-like seminomas and more differentiated non-seminomas. Based on the extraordinary sensitivity towards cisplatin-based chemotherapy, GCTs are recognized as a model of a curable disease, even in advanced, metastatic stages . HoweverEpigenetic modifications play a significant role in carcinogenesis and in response of cancer cells to chemotherapy through regulating the expression of genes involved in cell replication, tumor suppression, DNA repair and apoptosis . EpigeneKLF11, a putative tumour suppressor gene, and hypermethylation of CFLAR, a gene previously described in treatment resistance in GCTs.Chemical DNA demethylation can restore the expression of silenced genes, which can be achieved, for example, by the use of the first generation DNMT inhibitor (DNMTI) 5-azacitidine (5-aza), which is approved by the Food and Drug Administration (FDA) for the treatment of myelodysplastic syndromes. The cytotoxic activity of 5-aza as a ribonucleoside, which gets incorporated into both DNA and RNA, is based on broad effects on both DNA methylation patterns and the cell’s transcriptome and gene expression profile ,14. In aTP53, and 2102Ep, which has wild type TP53, in an in vitro model system of acquired cisplatin-resistance using isogenic, resistant sublines NCCIT-R and 2102Ep-R.Here, we examined the effect of 5-aza, which has not been examined in non-seminomatous GCT cells so far, on two distinct embryonal carcinoma (EC) cell lines, e.g., NCCIT bearing a mutant 50 values ranging from 18 to 23 nM  polymerase 1 (PARP1). The cisplatin sensitive EC cells treated with their respective IC50s of cisplatin for 72 h served as controls of apoptosis induction. In all four cell lines we detected a strong apoptotic response upon 72 h of treatment with the respective IC50 doses of 5-aza as single agent as evidenced by increased caspase-3 and PARP1 cleavage  or 2102Ep (0.8 µM) or a supra-lethal dose of 5 µM representing the IC50 of both resistant sublines NCCIT-R and 2102Ep-R. With respect to a possible impact of the timing of cisplatin exposure after 5-aza treatment, two different schedules were tested. Cisplatin was applied either 24 h after re-plating (immediate sequence), or after a 168 h drug-free period following 5-aza exposure (delayed sequence) to assess possible prolonged effects of 5-aza on cisplatin sensitivity  was significantly less effective in the isogenic cisplatin-resistant sublines with a mean relative viability of 38% vs. 63% (p ≤ 0.0001) for NCCIT/-R and 46% vs. 69% (p = 0.0007) for 2102Ep/-R. Moreover, cisplatin demonstrated a dose-dependent toxicity in the resistant sublines after 48 h of treatment with either the aforementioned doses or a supra-lethal dose, showing a 14% reduction of the mean viability for NCCIT-R (p = 0.0091) and a 30% for 2102Ep-R (p = 0.001)  b,c. Toge50 doses of 5-aza for all cell lines, decreased cell viability to an unexpectedly high extent compared to the experiments shown in Treatment with 10 nM 5-aza alone did not significantly impact cell viability when compared to untreated controls, confirming the results in 50 of the corresponding sensitive cell lines, ranging from 14% (p = 0.012) to 18% (p = 0.014) in NCCIT-R cells, and from 15% (p = 0.019) to a borderline significant 13% (p = 0.058) in 2102Ep-R cells for immediate and delayed sequencing, respectively. Importantly, combined treatment decreased cell viability in the resistant sublines almost to levels achieved by cisplatin alone in the sensitive cell lines (p = 0.21). Except for the cisplatin-resistant, TP53-mutant subclone NCCIT-R , combining 10nM of 5-aza with a supra-physiological dose of cisplatin (5µM) did not add a relevant cytotoxic effect compared to 5µM of cisplatin alone , which did not show significant impact on cell growth  are thought to be one mechanism leading to cisplatin resistance in GCTs, mediated by an altered apoptotic response to treatment [50 values for the sensitive lines NCCIT and 2102Ep. The IC50 of the resistant sublines was much higher at supra-physiological 5 µM as reported before, which thus appropriately reflects the clinical burden of cisplatin-resistant disease [Both cell lines used in this project originate from embryonal carcinomas, a common chemo-sensitive subtype of non-seminomatous GCTs. The cell line NCCIT is derived from a primary mediastinal germ cell tumor with p53-deficiency, as it is frequently found in these rare tumors. reatment ,18. Impo disease . Thus, sRASSF1A, HIC1 and MGMT promoters [Epigenetics refers to gene expression alterations without changes in the DNA sequence. The most intensively studied epigenetic mechanisms comprise DNA methylation, regulation by microRNAs, and covalent, post-translational histone tail modifications, i.e., methylation and acetylation . The difromoters ,11,12. Aberrant DNA hypermethylation is a reversible epigenetic alteration, which can be therapeutically targeted with the aim of achieving global DNA hypomethylation and re-expression of tumor-suppressor genes ,21. NANOG or SOX2, and Myc target genes in NT2/D1 cells [NANOG and OCT3/4, or hypermethylation of distinct putative drivers of cisplatin-resistance, i.e., CFLAR, together with decreased cisplatin resistance upon 5-aza treatment in the seminoma cell line TCam-2 [TP53-mutation status, which is why our results add another important aspect to the complexity by showing a sustained strong apoptotic response and a  restoration of cisplatin sensitivity irrespective of the cell lines TP53 mutational status . This observation supports the hypothesis that the effects of DNMT inhibition may be multi-facetted, which could mean that the majority of cisplatin-resistant GCTs may be amenable to 5-aza (and/or other DNMTI) treatment irrespective of key clinical aspects related to cisplatin-resistance, particularly p53 pathway aberrations [Overall, our results, where single agent 5-aza was equally effective in both p53-deficient (NCCIT) and proficient 2102Ep) isogenic in vitro models of cisplatin-resistant GCTs, are in line with those reported for the treatment of the embryonal carcinoma cell line NT2/D1 and its resistant subline NT2/D1-R1 with either decitabine or guadecitabine. Moreover, sensitivity towards therapeutic DNA demethylation with either decitabine or guadecitabine was functionally dependent on a high expression of DNMT3B ,21. This02Ep isogD1 cells ,23. On te TCam-2 . Taken trrations .In general, GCTs consistently displayed an extraordinary sensitivity towards DNA demethylating drugs at low nanomolar concentrations, which is why DNA demethylation, either alone or in combination with other cytotoxic substances, holds promise as a new treatment strategy in refractory GCTs.Being the first and clinically the most often used epigenetic drug so far, 5-aza is currently recommended as first-line treatment of high-risk myelodysplastic syndromes and acute myeloid leukemia in patients unsuitable for more aggressive chemotherapy. Repeated cycles of 5-aza have proven to be highly effective and well tolerated in such patients ,25,26. A2 daily, days 1 to 7, every 4 weeks, with a reported peak plasma concentration 750 ± 403 ng/mL occurring 0.5 h after infection with a half-life of 41 ± 8 min. A slightly different but also intense dosage was applied in the phase II trial by Roth et al.  evaluating 5-aza as a single agent in 17 refractory GCC patients, where no responses were seen [The standard dosage to treat hematological malignancies is a subcutaneous injection of 75 mg/mere seen . With re50 determined in EC cell lines in this study, as well as those reported for other DNMTIs in the literature, are much lower and more in the range found in hematological neoplastic cells than the doses causing anti-neoplastic effects in solid cancer models when using other entities, such as pancreatic, hepatocellular, or breast cancer cells [Hypothetically, much lower doses may thus be effective in vivo to  overcome cisplatin-resistance in GCTs and to reduce toxic side effects at the same time. Interestingly, the ICer cells ,30,31. LPresumably due to the multifactorial origin of platinum-resistance, targeted approaches using single agents failed to achieve clinically meaningful responses in refractory GCTs so far. Combined therapeutic approaches may thus be more promising . InteresThe extragonadal embryonal carcinoma cell line NCCIT was purchased from the American Type Culture Collection . The gonadal embryonal carcinoma cell line 2102Ep was a generous gift from P.W. Andrews . Cells were routinely checked for contamination with mycoplasma and inspected microscopically for stable phenotype. All the cell lines used in the current research have been tested and authenticated.L-Glutamine (2 mM) , and 50 IU/mL penicillin and 50 µg/mL Streptomycin . For both cell lines, isogenic, cisplatin-resistant subclones were developed by repeated exposure to sublethal, increasing cisplatin-doses as described before [2. Cells were passaged when reaching 80–90% confluency, and only used for assays in passages 3–7. Both cell lines were maintained in Dulbecco’s modified Eagle medium (DMEM)/F12, 1:1 nutrient mixture medium  with 10% fetal bovine serum supplement  plus d before ,35. Cell5-azacitidine (5-aza) was purchased from Selleckchem . For the treatment, 5-aza was freshly dissolved in phosphate-buffered saline (PBS) and added daily to the culturing medium at the indicated concentrations for 72 h with drug-containing media replenishment each day. Cisplatin solution was a generous gift from Medac Pharma . Cisplatin treatment was performed at the indicated concentrations and time points for 48 h. 5 cells/well were seeded in 6-well format in complete growth medium (2 mL/well). Following the plating, the cells were incubated overnight to allow proper adhesion before drug treatment. For determination of the IC50 for 5-aza, cells were treated with different doses of the drug in triplicate for 72 h. Cell viability was assessed by Trypan blue staining method combined with the automatic cell counting using Beckman Coulter Vi-CELL . For combined treatment, two approaches were applied: (i) cells were treated with 5-aza at either a fixed-dose of 10 nM, or the respective IC50 over 72 h. Cells were then washed with PBS, trypsinized, pelleted using centrifugation at 300× g, and resuspended in 6 mL complete growth medium. One quarter of the cell suspension volume was re-plated in a 6-well, incubated for 24 h, and treated with cisplatin at either the IC50 or a fixed-dose of 5 µM (IC50 of the resistant subclones NCCIT-R and 2102Ep-R) for another 48 h. (ii) In order to assess possible long-term effects of low-dose 5-aza, cells were treated in the same way except for a prolonged drug-free period of 7 days (168 h) between the end of 5-aza treatment and onset of cisplatin exposure. Cell viability was assessed by both the trypan blue staining assay and a semi-automated cell counting using a Beckman Coulter Vi-CELL . Each experiment was performed in triplicates and repeated independently at least two times.For assessment of cell viability, 1.5 × 103 cells of each cell line were treated with or without 5-aza and then analyzed by flow cytometry  after propidium iodide (PI) staining as described before [For cell cycle analysis, 5 × 10d before  Cells wev/v), 0,25% NaDesoxycholat (10%) (w/v), 1mM Phenylmethylsulfonylfluorid (PMSF), 1mM Natriumorthovanadat (Na3VO4), 10 mL Complete Protease Inhibitors (Aprotinin/Leupeptin)). Equal amounts of total protein (30μg) were electrophoresed using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), all as described before [Whole cell extracts were isolated using RIPA buffer . The statistical significance of differences in cell viability and in the cell cycle phases distribution was calculated using two-sided, unpaired student’s In summary, we report here that 5-aza not only effectively affects cell viability and strongly induces apoptosis at low nanomolar concentrations as a single agent, but also achieves a substantial re-sensitization towards cisplatin in an in vitro EC model of cisplatin-resistance supporting the evaluation of low-dose DNMTIs in cisplatin-resistant GCTs in the setting of a clinical study.


The globalization of problem-based learning (PBL) in health professions education has been both celebrated and criticized. Using a critical narrative review approach, underpinned by our archive of global PBL literature and a targeted literature search, we analyze these dominant global discourses of PBL in health professions education. More precisely, we explore what is missed when the globalization of PBL is theorized either as a positive consequence of standardization, or a problematic spread of Western educational ideals and values around the world. We make visible how two dominant global discourses, a universalist and culturalist discourse, have emerged in the global proliferation of PBL. We also discuss the limitations of the two discourses by demonstrating how they either ignore contextual and cultural diversity or see it as problematic. We then turn to a perspective that has been marginalized in the PBL literature that emphasizes the global origins of PBL, transcending the dichotomy between West and non-West. We make a case for relating to PBL as a plural construct in order to learn from the cultural and situational nuances of educational activities labeled PBL around the world. We argue that PBL as a singular and universal concept has no global future, yet versions of PBL may continue to thrive locally. Finally, we propose avenues for future research that may help elucidate the global and local values that underpin our curricula, as well as the socio-political factors that perpetuate neo-colonialist views and practices in the uptake and implementation of PBL approaches across the globe. The globalization of health professions education has resulted in more collaboration between educational institutions and a greater flux of students and teachers across the globe . In other words, individuals and organizations that embody dominant conversations related to PBL are regarded as legitimate authorities on how to practice PBL, and in turn contribute to the proliferation of these conversations. At least in health professions education, PBL has been a dominant conversation topic worldwide for 50 years, as evidenced by the number of citations related to PBL Azer . These cPrevious research on the globalization of health professions education distinguishes between discourses around universalism and homogenization  and discourses around culturalism and hybridization  . In practice, this enables institutions “who have historically been in a position to build capacity and experience in PBL and share their knowledge and research worldwide, such as Maastricht University, to continue being looked at for expertise (…), possibly at the expense of others who did not have such a head start or who have a different educational message that is less aligned with mainstream global discourse”  rather than educational effectiveness per se  and instead be shaped by the global dissemination of best practices, underpinned by research on the science of learning. PBL, as a term, has come to mean many different things. It is often used simply as a short form to signify an appreciation for the idea that learning can be self-directed and can move away from large group lectures. For this reason, each institution may find it useful to define whether their approach is reminiscent of PBL, how they interpret PBL, and how they shape and implement it locally. At a global level, institutions may share their experiences with other institutions as part of a global conversation for improving education. We believe this ultimate goal of improving education is best served if this conversation includes participants bringing in a rich diversity of educational approaches and perspectives, and focuses on how we can deliver high quality graduates rather than how we label our approach or who is allowed to use that label. This means recognizing and encouraging the plurality of approaches that has developed, not only as part of the PBL movement, but as part of student-centered and community-oriented education more broadly. To be effective, power relations must also be considered when organizing and participating in global sharing events and practices, considering that those currently in power have more access to resources, and greater capacity to control how we disseminate and share knowledge about PBL approaches.Thus, the term PBL as a singular and universal concept has no global future. Globally, we can only talk about PBL in the plural sense, which we may emphasize by using plural expressions such as ‘PBL approaches’ or ‘PBL strategies’. This subtle change in terminology has implications for how the term is currently used worldwide  because these situations often require a singular, shared understanding across contexts and institutions. Rather than “nailing down definitions”, the potential value of exploring the meaning of educational concepts, including the philosophies and values that underpin them in different contexts worldwide, has been emphasized previously in terms of opening up alternative ways of seeing and knowing what may be better in other contexts (Naidu and Kumagai In addition to functioning as a platform for sharing local experiences and interpretations of PBL, how and why it might function in different contexts, and opening up to approaches of student-centered education more broadly, research on PBL approaches may focus on the factors that draw institutions worldwide to constructs of PBL. Specifically, future research should analyze why people and institutions are drawn to PBL constructs, what they see as added value of using the term, how they take decisions regarding its adoption and implementation, and what forces guide these decisions. These avenues of research may help elucidate the global and local values that underpin our curricula, as well as the socio-political factors that perpetuate neo-colonialist views and practices in the uptake and implementation of PBL approaches across the globe.


Summary: IgA nephropathy is the most common type of primary glomerulonephritis worldwide. At least 25% of patients may progress to kidney failure requiring dialysis or transplantation. Treatment of IgA nephropathy using generalized immunosuppression is controversial, with concerns regarding the balance of safety and efficacy in a nonspecific approach. This review describes the recent scientific evidence, and a current clinical trial, investigating whether spleen tyrosine kinase (SYK) may be a novel and selective therapeutic target for IgA nephropathy. SYK, a cytoplasmic tyrosine kinase, has a pivotal role as an early intermediate in intracellular signal transduction cascades for the B-cell receptor and the immunoglobulin Fc receptor, and thus is critical for B-cell proliferation, differentiation, and activation, and for mediating proinflammatory responses after Fc-receptor engagement in various cell types. In renal biopsy specimens of patients with IgA nephropathy, increased expression and phosphorylation of SYK were detected, and this correlated with the histologic features of mesangial and endocapillary proliferation. In cell culture studies, patient-derived IgA1 stimulated mesangial cell SYK activation, cell proliferation, and cytokine production, and these responses were attenuated by pharmacologic or molecular inhibition of SYK. A global, randomized, double-blind, placebo-controlled trial investigating the safety and efficacy of fostamatinib  in the treatment of patients with IgA nephropathy is ongoing, which may provide important evidence of the safety and efficacy of targeting this pathway in clinical disease. Spleen tyrosine kinase (SYK) is an immunoreceptor-associated protein tyrosine kinase with numerous biological functions. First identified in 1991  domains and a C-terminal kinase domain A.3 The Slinker-kinase sandwich .26More recent attention has focused on the role of SYK in signaling for nonclassic immunoreceptors. SYK has been implicated in integrin signaling in myeloid cells, which is thought to be dependent on the association of integrins with ITAM-containing adapter proteins such as FcRγ chain and DAP12.Given its important role in immunoreceptor signaling and in mediating immune and inflammatory responses, SYK function has been studied in a number of in vivo models of allergic and autoimmune disease. Studies using bone marrow chimeras generated using SYK-deficient progenitor cells, for example, confirmed that SYK deficiency in the hematopoietic compartment conferred resistance to a passively transferred antibody-dependent model of inflammatory arthritis.20,A number of pharmaceutical companies are working to develop compounds to inhibit SYK for use in allergic and autoimmune disease.38Current understanding of IgA nephropathy proposes a multihit model of disease pathogenesis.Evidence for the latter phenomenon initially was provided by immunohistochemical analysis of renal biopsy specimens from patients with IgA nephropathy.The functional importance of SYK in IgA nephropathy initially was investigated using a cell culture model, wherein human glomerular mesangial cells were stimulated with IgA1 purified from patients with IgA nephropathy.The identity of the specific mesangial cell receptor(s) for IgA1 in patients with IgA nephropathy has been controversial, and a number of putative candidates have been suggested. The transferrin receptor (CD71) perhaps is the best characterized of these, being expressed on human mesangial cells, and up-regulated and co-localized with IgA1 deposits in IgA nephropathy.The specific role of SYK in myeloid cells expressing the classic FcαR1 (CD89) in the pathogenesis of IgA nephropathy is less well studied. Historical data have suggested that glomerular and tubulointerstitial mononuclear phagocytic cell infiltration is associated with a worse prognosis in IgA nephropathyA number of in vivo models of human IgA nephropathy have been described, including spontaneous, inducible, and genetically modified systems.Nevertheless, important lessons about the potential role of antibody-mediated inflammation have been learned in models of anti–glomerular basement membrane (GBM) antibody-mediated glomerulonephritis in rodents, which are reliably characterized by significant glomerular inflammation and progression to renal failure. In the passive-transfer model of anti-GBM glomerulonephritis in Wistar Kyoto rats (nephrotoxic nephritis), treatment with fostamatinib  was effective in reducing macrophage infiltration, fibrinoid necrosis, and crescent formation, with a reduction in proteinuria and serum creatinine.In the active immunization model of anti-GBM glomerulonephritis , disease is induced by immunization with the α3-chain of type IV collagen (the Goodpasture autoantigen), resulting in the development of autoantibody and crescentic glomerulonephritis. In this model, early treatment with fostamatinib reduced the production of autoantibody and the severity of glomerulonephritis.It is challenging to attribute the importance of a specific molecule or cytokine to the pathogenesis of glomerulonephritis, given that multiple complex, inter-related pathways may be involved. Taken together, however, the available preclinical data imply that SYK makes a significant contribution to the pathogenesis of IgA nephropathy, in particular via its role in mediating proinflammatory responses after the deposition of IgA1- and IgG-containing immune complexes in the renal mesangium. Emerging evidence suggests that the interaction of IgA1 with various receptors on mesangial cells results in SYK activation, although the potential of signaling via other receptors  has yet to be explored specifically in IgA nephropathy. In addition, the role of SYK in the production of abnormal IgA1, and the autoantibodies directed against it, by B cells and plasma cells, has not been explicitly studied in IgA nephropathy, although data from other experimental immune models have suggested this may be a potential benefit, and other B-cell–directed therapies are undergoing clinical evaluation in IgA nephropathy. It is possible that a reduction in disease severity observed in the animal models of glomerulonephritis may be achieved by selective reduction (rather than complete abrogation) of SYK activity on these multiple pathwaysConversely, some of the therapeutic effects that have been observed when using small-molecule inhibitors may be attributed to off-target effects. R406 (the active metabolite of fostamatinib) is a potent inhibitor of SYK, although with a significant off-target effect on Flt3 and vascular endothelial growth factor receptor.A proof-of-principle global clinical trial of fostamatinib was performed to study the efficacy and safety of fostamatinib in the treatment of patients with IgA nephropathy (NCT02112838). This was a randomized, double-blind, placebo-controlled, clinical trial that included a run-in period with optimized control of hypertension and proteinuria with angiotensin-converting enzyme inhibitor or angiotensin II receptor blocker for 90 days before randomization. The patients then were randomized to groups receiving fostamatinib 100 mg twice daily, 150 mg twice daily, or placebo for 24 weeks. This study completed global recruitment in February 2018, and results are expected later in 2018. Both functional  and histopathologic end points will be evaluated because enrolled patients will be offered a repeat renal biopsy at the end of the treatment period. The results of this clinical trial will be pivotal in understanding whether SYK inhibition may be a safe and effective treatment of IgA nephropathy.


Casearia capitellata, Baccaurea motleyana, Phyllanthus pulcher and Strobilanthus crispus), indigenous to Malaysia, were extracted in different solvents, sequentially. The obtained 28 extracts were evaluated for their in vitro anticancer properties, using the MTS assay, on four human cancer cell lines: colon (HT-29), breast (MCF-7), prostate (DU-145) and lung (H460) cancers. The best anticancer activity was observed for the ethyl acetate (EA) extract of Casearia capitellata leaves on MCF-7 cell lines with IC50 2.0 μg/mL and its methanolic (MeOH) extract showed an outstanding activity against lung cancer cell lines. Dichloromethane (DCM) extract of Phyllanthus pulcher aerial parts showed the highest anticancer activity against DU-145 cell lines, while significant activity was exhibited by DCM extract of Phyllanthus pulcher roots on colon cancer cell lines with IC50 value of 8.1 μg/mL. Total phenolic content (TPC) ranged over 1–40 mg gallic acid equivalents (GAE)/g. For all the samples, highest yields of phenolics were obtained for MeOH extracts. Among all the extracts analyzed, the MeOH extracts of Strobilanthus crispus leaves exhibited the highest TPC than other samples (p < 0.05). This study shows that the nature of phenol determines its anticaner activity and not the number of phenols present.Different parts of four edible medicinal plants ( Plant based drugs have a long history in both traditional and modern societies as herbal remedies or crude drugs, or as purified compounds approved by the Food and Drug Administration and similar regulatory agencies ,2,3. DruInvestigations about natural products have recently regained prominence with the rapidly increasing understanding of their biological significance and increasing recognition of the origin and function of their structural diversity.Cancer is a leading cause of death worldwide. Breast and prostate cancer are two of the most common malignancies and contribute significantly to the societal and economic burden of cancer. About 10 million new cases are diagnosed and over 6 million deaths occur worldwide annually (excluding non-melanoma skin cancers) . ImportaPhyllanthus pulcher (Euphorbiaceae), Casearia capitellata (Flacourtiaceae), Strobilanthes crispus (Acanthaceae) and Baccaurea motleyana (Phyllanthaceae) for their TPC and anticancer potential. Plants selected for this study were chosen because of their use in local traditional cuisine. Information on the traditional culinary purposes of these plants was collected through a structured literature review. The present paper deals with the screening of the following medicinal plants: P. pulcher from genus Phyllanthus is found throughout South East Asia and is locally  known as Naga Buana (Dragon of the World). The plant is used extensively in Malay traditional medicines: as a decoction for stomach ache, the leaves to treat tooth ache, poultices are applied to the nose for ulceration, to the skins to treat boils, and to the abdomen of children with kidney problems [C. capitellata from the family of Flacourtiaceae is a lowland rain forest treeendemic to Malaysia. Casearia species are traditionally used in Malaysia as antiseptic, cicatrizant and topical anasthetic agent [Strobilanthes is a genus of about 250 species of flowering plants in the family Acanthaceae, mostly native to tropical Asia, but with a few species extending north into temperate regions of Asia. Traditionally, the leaves of S. crispus were boiled with water and the filtrates were used in traditional medicine in Malaysia and Indonesia as antidiabetic, diuretic, antilytic, and laxative. S. crispus has been proven to be possessing high antioxidant and antihyperglycemic properties [Baccaurea motleyana (Family: Phyllanthaceae) is a species of fruit tree, which grows wild in parts of Southeast Asia and is cultivated for its fruit in Thailand and Peninsular Malaysia. The fruits are approximately 2 to 5 centimeters long and about too wide and grow in strands. Each fruit has velvety pinkish, yellow, or brown skin, which wrinkles at ripening and is filled with whitish pulp containing 3 to 5 seeds. The pulp is sweet to acidic in taste. The fruits are eaten fresh or processed into beverages and wine. The tree is also used for shade and low-quality wood [The study shows problems . C. capiic agent . They haic agent , antiinfic agent , antihypic agent , and hypic agent  activitiic agent ,20. Strooperties ,22. Baccity wood .A survey of the literature revealed that no studies on the anticancer potential of extracts from these plants have been undertaken on four human cancer cell lines: breast cancer cell line (MCF-7), prostate cancer cell line (DU-145), colon cancer cell line (HT-29) and lung cancer cell line (H460). It is known that different cell lines might exhibit different sensitivities towards an anticancer compound, so the use of more than one cell lines was therefore considered necessary in the evaluation of anticancer effects. Bearing this in mind, four human cell lines of different histological origin were used in the present study. The study is aimed to evaluate anticancer potential of sequentially prepared extracts from different parts of these plants on above said cell lines. The aim of this study was so to investigate the anticancer activity of these extracts against a panel of tumor cell lines. The TPC from these extracts was also determined. The findings of the study will be helpful in understanding anticancer and antioxidant potential of these plants and significance for the development of new anticancer drugs.i.e., 2.0 to 40%. However, the highest yield was obtained for methanolic extracts. It is in accordance with earlier observations [The yield of extracts, from different parts of all plants prepared sequentially in different solvents, is shown in rvations , reportiS. crispus leaves contained the highest amount of phenolics (40 mg/g) followed by P. pulcher roots (32 mg/g), C. apitellata (30.4 mg/g), B. motleyana fruit (21 mg/g), S. crispus flowers (17 mg/g), P. pulcher aerial parts (15 mg/g), whereas the lowest level was found in B. motleyana peel (12 mg/g) (S. crispus leaves (40 mg/g) as compared to S. crispus flowers (17 mg/g), while P. pulcher roots (32 mg/g) contained more phenols than P. pulcher aerial parts (15 mg/g). Whereas B. motleyana fruits (21 mg/g) contained more phenols in comparison to B. motleyana peel (12 mg/g).Total phenolic contents were determined spectrophotometrically using Folin-Ciocalteu reagent and results are presented in 12 mg/g) . Phenolsi.e., solvent employed. Having used several solvents, it was found that the best yields of phenolic compounds, for all the samples, were obtained in high polar medium, i.e., MeOH extracts. This is in agreement with a recent study on Pistacia vera, where it was found that the yield in total phenols depends on the method and the choice of solvent, and that the highest amount was obtained in highly polar extracts  [The variation among TPC may be assumed due to the presence of different types of phenols and other constituents in different parts/plants; selectively extractable depending upon the extraction medium,  powder) .The variations in amounts of phenolics in these plant extracts could be partly due to the differences in growing conditions. Under field conditions, the phenolic compositions of plant tissues are reported to be varying considerably with seasonal, genetic, and agronomic factors . In addi50 values) of plant extracts on the growth of MCF-7, HT-29, DU-145 and H460 cell lines, measured by MTS assay, are given in All the four cell lines, used in this assay, were capable of attachment to form a homogeneous monolayer on plastic substratum of culture wells, which is ideal for MTS assay. The MTS test is a simple bioassay used for the primary screening of crude plant extracts and isolated compounds. For each cell line, there was a linear relationship between cell number and absorbance; measured at 550 nm in both control and drug-treated wells. After 72 h of treatment, the anticancer activity of the plant extracts was determined. The anticancer activity . The different botanical taxa studied in this work are shown in Breast cancer (MCF-7), prostate cancer (DU-145), colon cancer (HT-29) and lung cancer (H460) cell lines were obtained from American Type Culture Collection . Sample absorbance was recorded by a Shimadzu UV-1700 pharma spectrophotometer at 765 nm.The plant material was subjected to dryness at room temperature, cut into small pieces and was then ground to a fine powder using an electric mill. The cold extraction method using organic solvents was employed to obtain crude extracts from all the plants. Dried powdered plant materials were sequentially extracted with hexane, dichloromethane (DCM), ethyl acetate (EA) and methanol (MeOH). The extraction process was conducted thrice to ensure the complete extraction of required materials and the combined filtrate, for individual sample, was concentrated using a rotary evaporator. Total phenolic contents (TPC) were determined using Folin-Ciocalteu reagent as described earlier  with sli2 atmosphere with 95% humidity. The optimal plating density of cell lines was determined to be 1 × 103 to ensure exponential growth throughout the experimental period and to ensure a linear relationship between absorbance at 550 nm and cell number when analyzed by the 3--5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) cytotoxicity assay to determine the anticancer effects of all plant extracts against a panel of cancer cell lines. The MTS cytotoxic assay has been considered a valuable tool in studying cytotoxicity induced by chemical agents, for many years. It uses a tetrazolium salt, which is reduced to a coloured formazan product by mitochondrial dehydrogenases in living cells. It is frequently used and is reported to be suitable for in vitro drug screening tests [5 cells per well and in a volume of 180 µL of culture media. The plates were incubated at 37 °C (5% CO2 and 95% air) overnight to allow attachment onto the wells, before the addition of plant extracts in replicates of 4 wells for each concentration to give final concentrations in the range of 0.1–100 µg/mL. Following incubation at 37 °C in an atmosphere of 5% CO2 and 95% air for 72 h, 50 µL of MTS (2 mg/mL in PBS) were added into each well containing the cells and medium above (blank). The plates were re-incubated for 1–4 h to allow metabolism of MTS by cellular mitochondrial dehydrogenases. The viable cells converted soluble yellow MTS into insoluble purple formazan. Following the incubation period, the medium containing MTS was aspirated from each well and 100 µL of DMSO was added to dissolve formazan in order to read the absorbance of soluble formazan at 550 nm, corresponding to number of living cells. The absorbance was determined at 550 nm using a 96-well microplate reader. The percentage of cell viability was calculated using the formula:Cells were grown in RPMI-1640 culture medium supplemented with 10% heat-inactivated foetal bovine serum, 100 IU/mL of penicillin and 100 μg/mL of streptomycin. Subconfluent cells were trypsinised using trypsin-EDTA after every 3–4 days and cells were placed in 3 mL of medium and syringed to obtain a suspension of single cells. The cell lines were maintained at 37 °C in 5% COng tests . Cells w50).Dose-response curves (% of viability Vs concentration) were constructed to obtain 50% inhibitory concentrations (ICp < 0.05. The inhibitory concentration 50% (IC50) was calculated from the dose–response curves  obtained by plotting the percentage of inhibition versus the concentrations.All the experiments were conducted in triplicate and statistical analyses was done according to SPSS 12.0. Data were expressed as means ± S.D. Statistical analysis was performed by one-way analysis of variance, followed by Duncan’s multiple range tests to determine the significance of differences among the means and multiple comparisons of unpaired data. Differences were considered significant at C. capitellaat, P. pulcher and S. crispus, can be an interesting source of anticancer compounds. Further studies identifying and isolating the key phenolic compounds may allow for a potential biomedical application in the therapy of cancer diseases.In conclusion, this work reveals that selected Malaysian flora species, including


Characterization studies revealed that all analogues of HW-155, as well as HW-155, were effective inhibitors of the fibril formation by hIAPP.The abnormal folding and aggregation of functional proteins into amyloid is a typical feature of many age-related diseases, including Type II diabetes. Growing evidence has revealed that the prevention of aggregate formation in culprit proteins could retard the progression of amyloid diseases. Human Amylin, also known as human islet amyloid polypeptide (hIAPP), is the major factor for categorizing Type II diabetes as an amyloid disease. Specifically, hIAPP has a great aggregation potential, which always results in a lethal situation for the pancreas. Many peptide inhibitors have been constructed from the various segments of the full-length hIAPP peptide; however, only a few have their origin from the screening of combinatorial peptidomimetic library. In this study, based on HW-155, which was previously discovered from a one–bead–one compound (OBOC) library to inhibit Aβ There is overwhelming evidence of the useful role of soluble amylin in glucose metabolism. However, the insoluble aggregates resulting from the misfolding of this protein are implicated in type II diabetes4. Just like the amyloid deposits formed in neurodegenerative disorders 5 and progressive diseases 7, amylin aggregates consist of stacked protofilaments, leading to a structural architecture known as the cross-β structure10. The oligomerization of amylin into amyloid fibrils has been suggested to be a stepwise process: first, the aggregation of monomers into colloidal spheres (nucleation units), which stop growing after reaching a threshold diameter; second, the association of the spheres to form linear chains of toxic, mature fibrils of pore-like morphology8. The extent of this oligomerization process is strongly correlated to pancreatic β-cell dysfunction and death in Type II diabetic patients13; therefore, an impairment of the initial monomer aggregation process is an appealing therapeutic approach to diabetes drug discovery and development.The human islet amyloid polypeptide (hIAPP), also known as amylin, is a unique amyloidogenic precursor peptide, and a critical pathogenic biomarker of Type II diabetes. Amylin is co-secreted with insulin by the β-cells of the pancreas, and co-stored in the secretory granules14. Hence, there is an urgent need for potent inhibitors of the process producing these pathogenic fibrils. In line with this, some inhibitors, including the peptides derived from the various secondary recognition elements along the amylin peptide chain17, engineered peptides20, constrained peptides27, small molecules29 and natural products32, have been reported. Many of these inhibitors are specific in their activity; however, a good number of them have exhibited dual inhibitory activity such that they can inhibit amyloid aggregation of multiple classes of proteins non-specifically35.To date, there is no cure for pancreatic amyloidosis, and there are only limited approved therapeutic strategies for its prevention37. γ-AApeptides contain N-acylated-N-aminoethyl amino acid units, and are derived from γ-PNAs38. Each monomeric unit of γ-AApeptides is equivalent to a dipeptide motif in a conventional α-peptide; thus a γ-AApeptide can project an identical number of functional groups as an α-peptide of the equal length. γ-AApeptides have been shown to form well-defined helical structures41, target bacterial membranes44, and specifically bind to protein surfaces to modulate protein functions and cellular activities48.In order to advance the application of peptidomimetics in chemical biology and drug discovery, we have recently introduced a new class of peptidomimetic scaffold called γ-AApeptides Fig. 36,37. γ-40 aggregation, HW-155, which was identified from an OBOC (one-bead-one-compound) γ-AApeptide combinatorial library  with little structural similarity to KLVFF, the core amyloid-forming unit of Aβ. Inspired by the findings on molecules that show cross-inhibition toward multiple amyloidosis35, and based on the fact that HW-155 was identified from an OBOC library, we hypothesized that HW-155 could be a non-specific amyloid inhibitor, and as such it may exhibit inhibitory activity toward the aggregation of hIAPP. To test our hypothesis, we synthesized HW-155 and eight (8) analogues (1–8) with methyl group substitution on each position , and investigated their ability to prevent the fibrillization of amylin (hIAPP), as well as to disrupt preformed hIAPP aggregates. Furthermore, we studied the aggregation behaviour and morphological characteristics of hIAPP in the presence and absence of various concentrations of HW-155 and its eight (8) analogues using transmission electron microscopy (TEM), time-dependent Thioflavin T (ThT) fluorescence assay, and atomic force microscopy (AFM). To further explore the biological potential of HW-155 and its analogues  alone . Using HW-155 as a lead sequence, we designed and performed a functional analysis of eight (8) analogues of HW-155, namely γ-AApeptides 1–8 , we substituted each of the N-side chains in HW-155 with an acetyl group, while alanine scanning was performed on the chiral side chains (5–8).Inspired by the preliminary data, we sought to understand the relationship between the structure of 1–8 Fig. . For fou1–8 was monitored by time-dependent ThT assay. In an initial attempt, we studied the anti-aggregation activity of 2.5-, and 10-fold molar excesses of 1–8 against hIAPP aggregation. Interestingly, peptides 2–8 were able to suppress the ThT fluorescence induced by hIAPP aggregation by about 50% or more, while, peptide 1 suppressed ThT fluorescence induced by hIAPP aggregation by at least 43%  in the presence of 0 µM in t3, 4, 5, and 6  improved the aggregation inhibitory activity. In the achiral side chain truncation series, peptide 4 emerged with the best anti-aggregation activity with almost 2-fold decrease in hIAPP aggregation across the concentrations tested compared to HW-155.Intriguingly, we identified that peptides  6 Table  could ine 5 Fig.  reduced HW-155 into methyl groups (5–8) gave rise to a better or similar inhibitory activity than HW-155. However, unlike acetylation, which seems to be more critical in inhibition near the N-terminal end, mutation to methyl side chain played a more important role near the C-terminal end, as evidenced by 5 and 6. As a matter of fact, for all the synthesized analogues, 5 exhibited the best anti-aggregation potential with almost 3–4 fold inhibition against hIAPP aggregation.Interestingly, conversion of the chiral side chain (derived from α-side chain) in 2, 3, 4, 5 and HW-155 with hIAPP.Given the promising inhibition study by ThT assay, we next carried out TEM experiment to further confirm the ability of the few most potent compounds to inhibit hIAPP aggregation. This is done by pre-incubating nearly equimolar amount of peptides 49. However, no such fibril was observed in the presence of 2, 3, 4, 5, and HW-155, indicating a significant prevention of fibril formation by hIAPP. A prevention of fibril formation by 2 and 5 resulted in monomers as observed in the TEM images. Likewise, oligomers seen in 3, 4, and HW-155 were a result of the inhibition of mature fibril formation.The TEM micrographs for hIAPP revealed a dense network of intertwined mature fibrils with linear, and thread-like morphology Fig. , indicat5 was able to disrupt preformed hIAPP fibrils at a tested concentration of 100 µM  h. Interestingly, only  µM Fig. .Figure 55 on hIAPP preformed aggregates, we allowed 10 µM hIAPP to age for 10 h before treatment with a 10–fold molar excess of 5. AFM showed a disappearance of mature fibrils . However, at a peptide concentration of 5 µM, peptides 3, 4, 5, and HW-155 reduced the cell viability by at least 10%, while peptide 2 showed a significant cell viability reduction of about 20%. At both concentrations tested, peptide 2 exhibited a mild cytotoxic effect on NIH-3T3 cells.Based on our findings of the inhibition of hIAPP fibrillization by lls Fig. . At 1 µM2, 3, 4, 5, and HW-155 to modulate the hIAPP amyloid–induced cytotoxicity, we treated NIH-3T3 cells with hIAPP (10 µM) and 5 µM peptide as described in the general information section. Results showed that cells treated with only hIAPP exhibited a significant reduction in survival, whereas treatment with 5 µM peptides 2, 3, 4, 5, and HW-155 was able to rescue the cells from the hIAPP amyloid cytotoxicity  capping reaction with 500 uL Acetic Acid. After capping, the beads were washed and alloc-deprotection reaction was done by reacting the beads with Pd (PPh3)4  and Me2NH BH3  (10 mins each time). The beads were then washed with DCM and DMF, followed by the reaction of the deprotected beads with acyl chloride  and DIPEA  in 3 mL DCM for 1 h or with the carboxylic acid , HOBt , and DIC  for 6 h (x2).Peptides were synthesized on the rink amide resin (0.6 mmol/g) on a Burrell Wrist-action shaker. 200 mg resin was treated twice (15 mins each time) with 3-mL 20% Piperidine in DMF to deprotect the Fmoc group. The beads were then washed twice with 3 mL each of DCM and DMF. The desired red Fig.  and are The above steps were repeated to assemble the desired sequence on the resin Fig. . After t50. 3.1 mg of hIAPP was dissolved in minimal amount of HFIP (50 uL) to remove aggregated hIAPP and the HFIP was removed over a gentle stream of Nitrogen gas. The process was done twice and 5 mL of Milli Q pure water was added to the disaggregated hIAPP film. The solution was then divided into 25 aliquots of 200 µL each. The 25 aliquots were lyophilized and reconstituted in 800 µL of PBS prior to use to obtain a hIAPP stock solution of 40 µM . hIAPP stock solution (40 µM) was also prepared as described above. The peptide, hIAPP and 15 µM ThT were added into a 96-well black plate. Equal volume of hIAPP solution was added into the 96-well black plate to make the final concentration of hIAPP in each well to be 10 µM. Time-dependent fluorescent change was monitored for 24 h by a Biotek Synergy H1 hybrid plate reader at excitation and emission wavelengths of 435 and 490 nm, respectively.Fi is the fluorescent intensity of the hIAPP treated with 5 at time t, Fh is the Fluorescent intensity of the hIAPP treated with PBS at time t.Similar to the protocol described above for the inhibition studies, hIAPP and ThT were added to PBS in a 96-well black plate and at concentrations of 10 and 15 µM, respectively. Time dependent fluorescent change was monitored for 10 h, after which 5 uL peptide was added to make a final peptide concentration of 100 µM. 5 µL PBS was also added for control wells. Each test well containing the peptide has a control well to which equal volume of PBS was added. The fluorescent change was then monitored for another 12 h. The relative Fluorescence, Fi/Fh was then plotted against the time (Hr),where Sample preparation was similar to ThT assay. Sample solution containing 10 µM hIAPP and 12.5 µM of the respective peptide were incubated at 30 °C for 24 h. 10 µL aliquot of the incubated solution was placed on a carbon-coated 200-mesh copper grid. After 10 mins, excess solution was wicked away and the grid was allowed to dry. This is then followed by the addition of 2% uranyl acetate solution (10 µL), which was allowed to float for 5 min. The excess solution was then removed using the blotting paper. The copper grid was left to dry at room temperature before imaging with a FEI Morgagni 268D TEM operated at 60 kV.A droplet (10 µL) of the sample solution was placed on a freshly cleaved mica disk and dried in open air at room temperature. Imaging was performed using a Multimode 8 AFM  and a Nanoscope V controller in the Peak-Force quantitative nanomechanics (QNM) mode (in air). A silicon cantilever probe with a spring constant of 2.8 N/m was used for imaging at room temperature (scan rate of ~1 Hz). The acquired images were leveled by subtracting a linear background.The percent aggregation was calculated using the formula below:Percent aggregation = (F in the presence of inhibitor/F in the absence of inhibitor) × 100%, where F is the average of the ThT signals  from 600 min onwards. These time points were chosen because the maximum ThT signals were observed at these points in the uninhibited hIAPP control experiment.4 cells/well. When the cells reached 60% confluence, the cell culture medium in each well was then replaced with 100 μL of cell growth medium containing peptides 2, 3, 4, 5, and HW-155 alone at concentrations of 1 or 5 µM peptide with 10 µM hIAPP.Mouse embryonic fibroblasts NIH-3T3 cell were purchased from Lifetechnoglies. The NIH-3T3 cell were grown at 37°C and 5% CO2 humidified atmosphere in DMEM medium, respectively, supplemented with 10% (v/v) heat-inactivated fetal calf serum, 2 mM glutamine, 100 units/ml penicillin, and 100 mg/ml streptomycin . Cell proliferation was determined using the CCK-8 cell counting kit (Sigma- 41 Aldrich). Cells were seeded in 96-well plates at 1 × 10After incubation for 24 h at 37 °C, the peptides were washed with PBS three times. Then, 10 μL of CCK-8 dye and 100 μL of DMEM cell culture medium were added to each well, and the cells were incubated for another 1.5 h at 37 °C. The absorbance at 450 nm was measured by a Synergy H1 Hybrid Reader . Untreated cells served as controls with 100% viability. The results are presented as the mean ± SD of three measurements.Supplementary materials.


Osteosarcoma, the most common bone malignancy with a peak incidence at adolescence, had no survival improvement since decades. Persistent problems are chemo-resistance and metastatic spread. We developed in-vitro osteosarcoma models resistant to chemotherapy and in-vivo bioluminescent orthotopic cell-derived-xenografts (CDX). Continuous increasing drug concentration cultures in-vitro resulted in five methotrexate (MTX)-resistant and one doxorubicin (DOXO)-resistant cell lines. Resistance persisted after drug removal except for MG-63. Different resistance mechanisms were identified, affecting drug transport and action mechanisms specific to methotrexate  for MTX-resistant lines, or a multi-drug phenomenon (PgP up-regulation) for HOS-R/DOXO. Differential analysis of copy number abnormalities (aCGH) and gene expression (RNAseq) revealed changes of several chromosomic regions translated at transcriptomic level depending on drug and cell line, as well as different pathways implicated in invasive and metastatic potential  and immunity (enrichment in HLA cluster genes in 6p21.3) in HOS-R/DOXO. Resistant-CDX models  injected intratibially into NSG mice behaved as their parental counterpart at primary tumor site; however, they exhibited a slower growth rate and lower metastatic spread, although they retained resistance and CGH main characteristics without drug pressure. These models represent valuable tools to explore resistance mechanisms and new therapies in osteosarcoma. Osteosarcoma is the first primary malignant bone tumor that predominantly occurs during adolescence ,2. StandThe aim of the study was to establish and characterize osteosarcoma models resistant to common chemotherapeutic agents and analyze their behavior comparatively to their parental counterparts in-vitro and in-vivo in a bone orthotopic setting.In order to establish resistant cell lines, a panel of six osteosarcoma cell lines was exposed to increasing concentrations of methotrexate (MTX) or doxorubicin (DOXO). Parental and resistant lines were assessed for their response to chemotherapy in terms of cell viability, migration ability, and resistance index. Acquired resistance to MTX developed in 5/6 lines (83%) up 1 µM for HOS, Saos-2, Saos-2-B and 143B after 3 months, and up to 0.03 µM after nine months exposure for MG-63 A. Half-mNo differences in morphology, growth rate or migration ability were detected between the MTX-resistant cell lines and their parental counterparts . In contWe then evaluated the stability of this acquired chemotherapy resistance by culturing resistant lines with or without the compound for at least nine weeks, and determined the resulting IC50 values. Under continuous drug pressure (Drug-ON) all lines exhibited high Resistance Index (RI) values for MTX (>37), while Drug-OFF cell lines exhibited variable behavior A. HOS-R/The possibility of a cross-resistance to other compounds was evaluated in resistant cell lines in Drug-ON and Drug-OFF culture conditions A,B. CellCross-resistance was not detected with any tested compound in the MTX-resistance lines Drug-ON or Drug-OFF, except for MG-63-R/MTX A. SurpriFor HOS-R/DOXO, cross-resistance was detected with ETOP (RI = 276), VCR (RI = 172), and MTX (RI = 12) B. The coBased on copy number abnormalities (CNA) by Oligonucleotide Comparative Genetic Hybridization Array (aCGH) and gene expression (GE) profiles by RNA sequencing (RNAseq), the resistant lines clustered according to their genetic background rather than the resistance mechanisms  Comparison of CNA-acquired changes of each MTX-resistant line to their respective parental counterpart showed common acquired CNA in MTX-resistant lines issued from similar genetic backgrounds , in regions which appeared to contain genes involved in MTX metabolism and resistance. We further analyzed the differential expression of these genes by RNAseq between each parental and corresponding MTX-resistant line Drug-ON and Drug-OFF and validated some findings (dihydrofolate reductase (DHFR) and reduced folate carrier (RFC/SLC19A1)) at protein level A–C.Chromosome 5q region containing Dihydrofolate reductase (DHFR)  was gained in HOS-R/MTX and 143B-R/MTX, amplified in MG-63-R/MTX, and not modified in the other lines compared to their parental counterpart . While up value 1,60×10-15). In Drug-OFF conditions compared to parental lines, the most significant enriched pathway was histone acetyltransferase . Among the 100 most significant fold changes, we found genes implicated in osteosarcoma oncogenesis (RTN1) [To explore other potential mechanisms of resistance common to all MTX-resistant lines, we performed a differential GE analysis between all MTX-resistant lines Drug-ON versus their parental counterpart, and Drug-OFF versus their parental counterparts . DiffereHAT; ID120435. Amos (RTN1) , down-res (RTN1) ,10. Enris (RTN1) .p value 6.63×10-100) followed by MMP13 . Several other regions with acquired CNA containing multiple multidrug resistance genes were also up-regulated in the resistant line in GE  and has been implicated in osteosarcoma oncogenesis [p value 6,45×10-04), while none of the cancer stem cell markers  [In the unique doxorubicin-resistant line HOS-R/DOXO, the acquired gained CNA translated in an increased mRNA GE level . The mosne in GE C. Chr7q2ogenesis ,13. Decrd ABCG2)  were modThese drug-induced changes suggest a broader impact of the acquired resistance in tumor cell behavior, especially in an in-vivo context.To explore the impact of the in-vitro acquired resistance on the in-vivo tumor behavior, the parental and resistant HOS and Saos-2-B lines stably transduced with Luc/mKate2 vector to express luciferase (above 90% Luc/mKate2 positive cells)  were injBone engraftment rates of HOS-R/MTX and parental counterpart were maximal (100%), whereas the HOS-R/DOXO-CDX had a slightly lower engraftment rate (66%) .The HOS-resistant lines had more difficulties to adapt to the in-vivo bone environment, with an initial decrease in in-vivo bioluminescence (up to 27 days), followed by a slightly faster growth compared to the HOS-parental-CDX A.Bone engraftment rate of Saos-2-B-R/MTX and parental-CDX were comparable  . Both paThe different models revealed tumor-bearing tibia bone structure abnormalities similar to those observed in osteosarcoma patients.The resistant models retained the primary tumor-induced bone abnormalities of their parental counterpart in the Computed Tomography (CT scan) B. The slMetastatic foci were detected by in-vivo bioluminescence in all CDX models as early as 30 days after intratibial injection A except After mice sacrifice, cells isolated from primary tumors of each CDX model (no in-vivo treatment was performed) were cultured for two in-vitro passages. All resistant-CDX-derived cells grew in-vitro. Drug response was assessed. RI of in-vitro resistant-CDX-derived cells were either lower , higher , or stable  compared to the matched RI of the in-vitro initial resistant cells .Resistance to chemotherapy and metastatic phenotypes are the two main problems in osteosarcoma patients that lead to recurrence and death. We developed six new in-vitro osteosarcoma models, resistant either to MTX  or DOXO (HOS), by continuous in-vitro drug exposure, adding new models to those previously described ,18,19,20High level of in-vitro acquired resistance was obtained with MTX (5/6 cell lines), while only one of the six lines developed resistance to DOXO. This is consistent with observation in other models derived from patient samples which showed resistance to MTX while still sensitive to DOXO and CISP . ContinuFor the unique, highly DOXO-resistant line HOS-R/DOXO line RI = 224), we observed a multi-drug resistant phenotype, with cross-resistance to other agents used in osteosarcoma treatment and substrates of PgP , associated with ABCB1/MDR1 gain translated in high MDR1 mRNA and PgP protein expression. The DOXO-resistant line gained several chromosomic regions associated with mRNA expression up-regulation on the chromosome 7 region containing ABCB1/MDR1 and ABCB4, and several other regions encoding various multiple multi-drug resistance genes or genes implicated in the apoptotic response to doxorubicin. Up-regulation of MDR1 has been associated with chemo-resistance development of osteosarcoma tumor-initiating cells 4, we obs,24,25. AFor our MTX-resistant lines, high level of resistance was obtained (RI > 37) and persisted after drug removal although at lower level, except for MG-63 which lost MTX-resistance. Higher resistance has been observed in patient-derived cell lines . All ourSeveral more general cellular and biological pathways were modulated in the chemo-resistant lines related to cell adhesion/motility, extracellular matrix organization/degradation/composition, and cellular microenvironment. These processes, not fully assessed in-vitro, might affect the resistant cell behavior in-vivo. In DOXO- and MTX-resistant osteosarcoma cells, Fas expression was increased, suggesting a decreased in-vivo metastatic potential . SimilarWe developed in-vivo orthotopic intratibial bioluminescent parental and resistant-CDX models in NSG mice, with the experimental procedure used previously . The difOur resistant-CDX models can substantially help to evaluate new drug efficacy in osteosarcoma and complement the few pre-existing osteosarcoma models. Future work will include the comparison of our models to the PDX models that we are currently generating issued from relapsed osteosarcoma and the human relapsed osteosarcoma cohorts that might be available in the future .v/v) fetal bovine serum  at 37 °C in a humidified atmosphere (5% CO2 and 95% air), under mycoplasma free conditions.Human osteosarcoma cell lines HOS, 143B, Saos-2, Saos-2-B, MG-63 and IOR/OS18  with difDoxorubicin (DOXO), methotrexate (MTX), cisplatin (CISP), etoposide (ETOP), vincristine (VCR), and verapamil (VER) were purchased from Sigma Aldrich , mafosfamide (MAF) from Toronto Research Chemicals Inc. , and cabozantinib (Cabo) from LC Laboratories . All compounds were solubilized in dimethyl sulfoxide , except cisplatin, solubilized in N, N-dimethylformamide , at 10 mM stock solutions, and stored at −20 °C.Cells were seeded into 6-well plate at 100,000 cells/well  or 120,000 cells/well  in DMEM supplemented with 10% FBS, then exposed continuously to an initial concentration of 0.01 µM DOXO or 0.07 µM MTX. In parallel, vehicle (DMSO) treated cells  were seeded at the same conditions and used as controls. The medium was changed twice a week, and passages (1:10) were performed when cells had reached 80% confluence. The MTX concentration was progressively increased up to 0.3 µM for MG-63 and 1 µM for all other lines; DOXO concentration was raised up to 1.3 µM for HOS. The resistant lines were maintained in culture with the maximal tolerated drug concentration and named as “Drug-ON” cell lines. Drug pressure was stopped after resistance confirmation, and resistant cells were cultured in drug free medium for minimum nine weeks, designed as “Drug-OFF” cell lines. The same experiments were performed with MAF without success .The cell lines that did not developed resistance, died under drug continuous exposure. Therefore, no experiments could be done.Parental and resistant derived HOS, 143B, MG-63 and IOR/OS18 lines were seeded at 5000 cells/well, and cells derived from Saos-2 and Saos-2-B at 10,000 cells/well in a 96-well plate in DMEM supplemented with 10% FBS for both assays.Cell proliferation rate and doubling time were assessed using the IncuCyte live-cell imaging system . Phase-contrast photographs were collected for 72 h.The day after seeding, cells were incubated in the presence of a range of drug concentrations for 72 h . Verapamil was used at 5 μmol/L and cabozantinib at 0.1 μmol/L, to revert PgP function. Cell viability was evaluated using the CellTiter 96 Aqueous One Solution Cell Proliferation Assay (MTS assay) , according to the manufacturer instructions. The half-maximal inhibitory concentration (IC50) was determined using the GraphPad Prism5 software .Cells were seeded at 10,000 cells/well , or at 20,000 cells/well  in 96-well ImageLock tissue culture plates  in DMEM supplemented with 10% FBS. The day after, cell layers were scratched with the WoundMaker™, washed once and then incubated in the presence of the indicated drugs  or DMSO (control). The wound-healing was monitored using IncuCyte™ system for 48 h. Data analyses were performed using Graphpad Prism5 Software.Parental and resistant HOS and Saos-2-B cells were stably transduced with Luc/mKate2 vector as previously described .Animal experiments were approved by the CEEA26, Ethics Committee and the French Ministry of Research (APAFIS#1648-2015090713516480) and performed under the conditions established by the European Community (Directive 2010/63/UE).6 cells in matrigel solution at 4 mg/mL), as previously described [The parental cell lines  and their resistant counterparts to either MTX  or DOXO (HOS-Luc/mKate2/DOXO) were established into 7-week-old immunodeficient NSG mice by unilateral intratibial injection  in DMEM supplemented with 10% FBS.In-vivo and ex-vivo images were acquired using IVIS SpectrumCT  as previously described . BrieflyFormaldehyde-fixed-paraffin-embedded (FFPE) tissue sections (4 µm) were either stained with hematoxylin-eosin-safranin (HES) for morphology, or processed for immunohistochemistry as described before . PrimaryDNA and RNA from cells and CDX samples were isolated using AllPrep DNA/RNA mini kit (Qiagen) according to manufacturer’s instructions. Quantification/qualification were performed using Nanodrop 2000 spectrophotometer  and Bioanalyzer DNA 7500 .http://www.agilent.com). 500 ng of tumor and reference DNAs were fragmented with AluI and RsaI  and labeled with Cy5-dUTP and Cy3-dUTP, respectively. Hybridization was carried out on SurePrint G3 Human CGH Microarray 4 × 180K  arrays for 24 h at 65 °C in a rotating oven  at 20rpm and followed by appropriate washing steps. Microarrays were scanned with an Agilent G2505C DNA Microarray scanner at 100% PMT with 3 µm resolution at 20 °C in low ozone concentration environment. Data were extracted using the Feature Extraction software , along with protocol CGH_1105_Oct12. All further data manipulations were performed under the R statistical environment . Raw intensities were normalized according to their dye composition (Cy3 fitted over Cy5). Data were transformed to log2(Test/Ref) and normalized according to their local GC content through a lows regression. Resulting profiles were segmented with the CBS algorithm [Sex-matched normal DNA from a pooled human DNA  was used as a reference. Oligonucleotide aCGH processing was performed according to manufacturer’s protocol version 7.5 (https://www.bioinformatics.babraham.ac.uk/projects/fastqc/). Reads were mapped with Salmon v0.8.1 [RNAseq analysis was performed as previously described . RNAseq n v0.8.1  using GRn v0.8.1 .http://bioinfogp.cnb.csic.es/tools/venny/). Toppfun website was used for functional enrichment analysis (https://toppgene.cchmc.org/enrichment.jsp).Differential mRNA expression lists were compared using Venny diagram produced by Venny 2.1.0  was reversely transcribed into cDNA using M-MLV Reverse Transcriptase , according to the manufacturer’s recommendations. Amplifications monitored with StepOnePlus PCR System , were performed using Maxima SYBR Green/ROX qPCR kit , and with a first step at 95 °C for 10 min followed by 40 cycles with 95 °C for 15 s and 60 °C for 1min. Melting curve was performed at the end of the PCR  to identify unique PCR products. The sequences of the specific primers are described in Parental and resistant Drug-ON and Drug-OFF cell lines were seeded into 100*20mm culture dish at 130,000 cell/dish in DMEM supplemented with 10% FBS. Cells were collected at 80–90% confluence, followed by a PBS wash and re-suspended in lysis buffer  and stored at −20 °C. Proteins were extracted and measured with Pierce BCA Protein Assay Kit .Western-blot analysis was performed using specific primary antibody against topoisomerase-II alpha/beta (TOPO2) , anti-dihydrofolate reductase (DHFR) (1:1000), anti-reduced folate carrier (RFC) (1/200), anti-methylenetetrahydrofolate reductase (MTHFR) (1/1000), all purchased from abcam  and β-Actin (13E5) Rabbit mAb . Appropriate secondary antibodies  at 1:5000 dilution were used, followed by visualization with the enhanced chemiluminescence ECL reagent  and imaged with ChemiDoc™MP image system . Signal intensities were quantified with the Image Lab version5 .®5 Software . The one-way ANOVA analysis was used to compare the groups. p < 0.05 was considered to indicate a statistically significant difference.Data were shown as the mean ± standard error of mean (SEM) of three independent experiments performed using Graphpad PrismIn-vitro acquired resistance to MTX and DOXO, induced CNA and possible epigenetic changes that involved specific but also more general mechanisms of resistance that might influence OS cell behavior in their microenvironment. These resistant-CDX models can help to evaluate new drug efficacy in osteosarcoma and complement the few pre-existing osteosarcoma models ,18,19,20


Furthermore, XN, DXN, and TXN induce extensive apoptosis in all these carcinoma cell lines. Finally, TXN induces G0/G1 cell cycle arrest in the colon carcinoma cell line HT29. Our findings suggest that DXN and TXN could show promise as therapeutic agents against colorectal and liver cancer in preclinical studies without the drawback of metabolism into a phytoestrogen.Xanthohumol (XN), a prenylated flavonoid found in hops, inhibits growth in a variety of cancer cell lines; however, its use raises concerns as gut microbiota and the host’s hepatic cytochrome P450 enzymes metabolize it into the most potent phytoestrogen known, 8-prenylnaringenin (8-PN). The XN derivatives dihydroxanthohumol (DXN) and tetrahydroxanthohumol (TXN) are not metabolized into 8-PN and they show higher tissue concentrations in vivo compared with XN when orally administered to mice at the same dose. Here we show that DXN and TXN possess improved anti-proliferative activity compared with XN in two colon  and two hepatocellular  carcinoma cell lines, as indicated by their respective IC Humulus lupulus [First discovered at the turn of the twentieth century by Power et al., XN is a simple prenylated flavonoid found in the female inflorescences, or hop cones, of the hop plant  lupulus . Hops ar lupulus ,6,7,8,9. lupulus .2-extracted hops, a byproduct of the hops industry, which provides a less expensive alternative to synthesis [Synthesis of XN from phloracetophenone is inefficient ; therefoynthesis . Furtherynthesis . In humaynthesis .Eubacterium limosum metabolizes IX via O-demethylation , which is the first step in the metabolism of XN  than XN in these cell types. While XN and DXN do not appear to induce cell cycle arrest, HT29 cells treated with TXN arrest in the G0/G1 phase of the cell cycle. These findings demonstrate that the two non-estrogenic XN derivatives are attractive alternatives to test along with XN in future preclinical studies using mouse models of colorectal and hepatocellular carcinoma.Here we demonstrate the anti-proliferative and cytotoxic effects of XN in two human colon adenocarcinoma cell lines (HT29 and HCT116) and two human hepatocellular carcinoma cell lines (HepG2 and Huh7). Furthermore, we show for the first time that DXN and TXN possess similar effects, and in most cases, a lower half-maximal growth inhibitory concentration (IC50) of DXN and TXN for two colon carcinoma and two hepatocellular carcinoma cell lines was determined using the SRB colorimetric assay for cytotoxicity screening, as previously described [50 values experimentally determined for XN, DXN, and TXN in the colon carcinoma cell lines HCT116 and HT29, and the hepatocellular carcinoma cell lines HepG2 and Huh7. DXN and TXN (except HepG2) exhibited lower IC50 values than XN. For the cells most sensitive to DXN and TXN, the decrease in IC50 values was approximately 30% compared to XN  to XN treatment than HCT116, but showed a similar sensitivity to the derivatives DXN and TXN as HCT116  7-AAD(−)), indicating caspase activation without membrane alterations (early- to mid-stage apoptosis). In addition, there was a statistically significant increase in doubly stained SR-VAD-FMK(+) 7-AAD(+) cells, indicating cells in late apoptosis , while TXN induced slightly greater apoptosis (+7.1%) than XN .1 phase . The human liver carcinoma cell line HepG2 was provided by Dr. Tori Hagen . We purchased Huh7 from the American Type Tissue Collection . The other cancer cell lines were originally purchased from ATCC. All cancer cell lines were grown and maintained in Dulbecco’s Modified Eagle’s Medium (DMEM) without sodium pyruvate  supplemented with 10% fetal bovine serum  and 1% penicillin–streptomycin . All cell types were maintained in 5% COXN and TXN were provided by Hopsteiner Inc., while DXN was synthesized as described previously . The purCells were seeded into 96-well plates . After 24 h, cells were treated with either XN, DXN, or TXN, as described above. Cell viability was evaluated using a sulforhodamine B (SRB) assay , as described previously . AbsorbaAt various times after treatment with XN, DXN, or TXN, adherent and floating cells were collected for analysis. Apoptosis was assessed using flow cytometry-based annexin V  and a multicaspase assay kit . The annexin V assay is based on measurement of externalization of phosphatidyl serine, a common characteristic of cells undergoing apoptosis. The multicaspase assay is based on measurement of caspase enzymes activated during apoptosis. Cells were trypsinized, washed in Dulbecco’s Phosphate Buffered Saline (D-PBS), and stained with Annexin V, sulforhodamine-valyl-alanyl-aspartyl-fluoromethyl-ketone (SR-VAD-FMK), and 7-amino-actinomycin D (7AAD) according to the manufacturer’s instructions. Cell populations were quantified using a Guava personal cytometer .g, washed with PBS, and re-suspended in cellular DNA staining solution containing 40 mg/mL propidium iodide  and 100 mg/mL RNase A (Sigma-Aldrich) in PBS. After a 15-min incubation at room temperature, cell populations were quantified using a Guava personal cytometer (Guava Technologies). At various time points after treatment with XN, DXN, or TXN, adherent and floating cells were collected and fixed in 70% ethanol at one million cells per aliquot. Samples were centrifuged at 500× p ≤ 0.05.Statistical analyses were performed with the GraphPad Prism 7 software . Group differences were assessed with a one-way ANOVA, followed by a Sidak’s post hoc test. The Sidak’s post hoc test was used to compare every mean with either the control mean, or XN mean. Statistical significance was set at


Gallbladder cancer is the most common malignant neoplasm of the biliary tract, responsible for 80–95% of cases. Appropriate models are required for investigating the molecular pathogenesis of gallbladder cancer.In this study, we aimed to establish a gallbladder cancer cell line from primary tumour. Single cell RNA sequencing, Light and electron microscopy, DNA content analysis, cytogenetic analysis, short tandem repeat (STR) DNA fingerprint analysis, immunophenotypic characterization, and xeno-transplantation were utilized to characterize the novel ZJU-0430 cell line in vitro and in vivo.The cell line showed multiple cell shapes and characteristic epithelial morphologies under the microscope, but no too much heterogeneity by scRNA-Seq, with a population doubling time (PDT) of 19.81 h, which was shorter than that for GBC-SD cells. An immunophenotypic analysis revealed that ZJU-0430 cells were positive for CD24, CD44, CD29 and CD133 expression, and partially positive for CD184, and CD326 expression, and negative for CD34, CD90, CD117, and CD338 expression, similar to the primary cancer cells. A pathological analysis confirmed the origination of cell line from gallbladder tumour. ZJU-0430 cells had higher migration, invasion and proliferation properties than GBC-SD cells in vitro, and showed in vivo tumorigenicity in nude mouse xenograft settings.The results confirm the potential utility of ZJU-0430 cell line as a representative model of gallbladder cancer and suggest that it could be used in the in vitro and in vivo studies of gallbladder cancer pathogenesis and to develop new therapeutics.The online version of this article (10.1186/s12935-019-0911-1) contains supplementary material, which is available to authorized users. Gallbladder carcinoma (GBC) is an invasive adenocarcinoma that originates from the epithelial linking of the digestive system . GBC is Despite the progress in therapeutic strategies, the overall survival rate has remained poor, mainly due to late diagnosis, early metastasis, ineffective surgical resection, and insensitivity to chemoradiation , 6–8. ThIn this study, a novel gallbladder cell line derived from a primary GBC, referred to as ZJU-0430, was successfully established. All our data together confirmed that it as a potentially useful model for the further study of this disease.This study was performed in accordance with the Declaration of Helsinki of 1975, and the official recommendations of Chinese Community Guidelines, and was approved by the Ethics Committee and Institutional Review Board of the Sir Run Run Hospital. Written informed consent was obtained from the patient.A 74-year-old male patient with pain in the upper abdomen was admitted in our centre. Gastroscopy showed the presence of a gastric antral ulcer (stage S2), and irregular deep concave ulcer at the gastric angle, and cancer priority. Abdominal computed tomography (CT) also found a 1.5-cm thick wall at the bottom of the gallbladder. A radioimmunoassay showed that the patient’s serum levels of a variety of biomarkers were normal  except for ferritin, which was high . A radical resection of stomach and gallbladder were performed and a pathological examination showed that gastric carcinoma was a poorly differentiated adenocarcinoma derived from signet-ring cell carcinoma, whereas the primary gallbladder cancer was a well differentiated adenocarcinoma.GBC-SD cells were obtained from the Cell Bank of Type Culture Collection of Chinese Academy of Sciences , and maintained in Roswell Park Memorial Institute (RPMI) 1640 medium  supplemented by 10% fetal bovine serum (FBS) , and 1% penicillin/streptomycin and amphotericin B (Invitrogen).3), and transferred into a gentleMACS™ C tube containing the buffer from the Tumor Dissociation Kit (human), and subjected to a gentleMACS™ Tissue Dissociator. Cells were cultured in complete growth RPMI 1640 medium (containing additives) at 37 °C in a humidified incubator containing an atmosphere of 5% CO2. Fibroblasts were removed by differential trypsinisation as described previously   assays were performed. As shown in Fig. A colony formation was also performed to examine the ability of ZJU-0430 cells proliferate. As shown in Fig. In order to investigate their in vivo tumorigenic capacity, ZJU-0430 cell suspensions were injected subcutaneously into the flank of nude mice. Tumour development was observed in all of mice and no obvious metastatic lesions were observed. Gross examination revealed that the tumours were round or oval shaped, and often showed mucus production. The atypia of tumour cells was obvious, when arranged with a small strip of cord and viewed under a microscopy they formed different cavities that included foam cells, cancer cells and normal epithelial cells Fig. d, e.To characterize the original ZJU-0430 cell lines, the original GBC, and the original gastric cancer, a series of markers were examined by IHC technology. As shown in Fig. To interpret the characteristic observed during the experimental investigation, we do single cell RNA sequencing analysis on ZJU-0430 cell line. By using Seurat pipeline, we identified 3 main cell clusters Fig. b. All thHuman GBC is an extremely malignant disease owning to its relative rarity, histological heterogeneity, and proximity to vital structures. Surgical resection, and chemoradiotherapy are ineffective , 8, 40, Cell lines derived from primary tumours are more likely to reflect the characteristics of the original primary tumour more accurately. Based on this idea, here we report the establishment of a GBC cell line designated as ZJU-0430, which was derived from a 74-year-old male patient who had some serum ferritin abnormalities, and GBC that was a well-differentiated adenocarcinoma. The cell grew in adherent monolayer pattern with at least two morphologies that co-existing. scRNA-Seq results revealed that ZJU-0430 cell line was no too much heterogeneity although the morphological difference. Optical microscopy, ultrastructural examination, immunostaining of epithelial markers for positive  and negative mesenchymal expression (vimentin), and H&E staining of cells displayed features that were typical of malignant epithelial cells. The STR genotyping analysis showed the ZJU-0430 cell line was of human origin and that it differed from other established cell lines. A mycoplasma examination confirmed ZJU-0430 to be free of mycoplasma. The PDT was approximately 19.81 h, which was shorter than GBC-SD cells. Taken together, these results suggest that ZJU-0430 cell line is a representative of the disease and may be used for pre-clinical investigation of the pathogenesis and treatment of GBC.Recently, Devendra Chaudhary, et al. reported that 46.7% of GBCs have an abnormal karyotype . In the Mycoplasma can impede a whole range of cellular properties and functions and hinders the use of cells and their derived products in pharmacological area . The culAlthough numerous GBC cell lines have been documented, including G-415, NOZ, KMG-A, GBK-1, FU-GBC-1, FU-GBC-2, PTHrP-GBK, GB-d1, TGBC1TKB, TGBC2TKB, OCUG-1, TUGBK-1, HAG-1, GBC-SD, SGC-996, EH-GB1, EH-GB2 and TJ-GBC2 , 47, 48,GBC is highly aggressive in patients ; therefoAn immunoblotting assay showed ZJU-0430 cells to be positive for biliary tract cancer epithelial marker , which are found to be positive at high rates in real GBC specimens. The mesenchymal marker vimentin was also tested, but it is not found to be expressed. Since the patient (source of ZJU-0430) had been diagnosed with gastric carcinoma before GBC, gastric carcinoma markers were also explored on the xenograft tumour. The gastric carcinoma markers MUC2, MUC5AC, and MUC6 were expressed weakly or not at all. Since the colon and liver are contiguous with each other, the origin of ZJU-0430 cell was a little unclear. In this study, liver markers  and colon markers  were used for staining of xenograft-derived tumours, with the result that all markers were either expressed only weakly, or were not express at all. In addition, the colon cancer marker CDX2 protein were also not found to be expressed on original GBC. Therefore, ZJU-0430 cell line was verified to be originated from the gallbladder primary site.We have successfully established a human GBC cell line referred to as ZJU-0430 that is identical to the original surgical specimen. This well-characterized GBC cell line should be helpful in the investigation of the pathogenesis of GBC, and may help in developing early diagnostic tools as well as potential targeted therapies.Additional file 1. STR Profile Report for ZJU-0430 at P100.Additional file 2. STR Profile Report for ZJU-0430 at P0.Additional file 3: Figure S1. Immunophenotypic analysis for ZJU-0430 as determined by flow cytometry. The representative graph of CD24, CD44, CD29, CD34, CD90, CD117, CD133, CD184 CD326, CD338 protein expression were detected on primary cells and P100 cells of ZJU-0430.Additional file 4: Figure S2. IHC for original gastric cancer sections. ZJU-0430 original gastric cancer tissues positively expressed most gastrointestinal tract markers , and negative expressed β-catenin and SATB2.Additional file 5: Figure S3. Characteristic of ZJU-0430 cell line in by scRNA-Seq data. (a) tSNE plot of ZJU-0430 cell line clusters defined by Seurat pipline before merging similar sub cell clusters. (b) UMAP plot of ZJU-0430 cell line clusters as showed in (a). (c) Dot heatmap of CD24, CD44, CD29, CD133 expression in each cell clusters. Heatmap showed the expression pattern of genes associated with Wnt signaling pathway (d), extracellular exosome (e), apoptotic signaling pathway (f) and nuclear transcribed mRNA catabolic process nonsense mediated (g).