Methotrexate Resistance Research Articles

Activation of RSK2 upregulates SOX8 to promote methotrexate resistance in gestational trophoblastic neoplasia

Resistance to chemotherapy is frequently driven by aberrantly activated kinases in cancer. Herein, we characterized the global phosphoproteomic alterations associated with methotrexate (MTX) resistance in gestational trophoblastic neoplastic (GTN) cells. A total of 1111 phosphosites on 713 proteins were significantly changed, with highly elevated Ribosomal S6 Kinase 2 (RSK2) phosphorylation (pS227) observed in MTX-resistant GTN cells. Activation of RSK2 promoted cell proliferation and survival after MTX treatment in GTN cell models. Interestingly, RSK2 might play an important role in the regulation of reactive oxygen species (ROS) homeostasis, as manipulation of RSK2 activation affected ROS accumulation and SOX8 expression in GTN cells. In addition, overexpression of SOX8 partly rescued cell proliferation and survival in RSK2-depleted MTX-resistant GTN cells, suggesting that SOX8 might serve as a downstream effector of RSK2 to promote MTX resistance in GTN cells. Highly activated RSK2/SOX8 signaling was observed in MTX-resistant GTN specimens. Further, the RSK2 inhibitor BIX02565 effectively reduced SOX8 expression, induced ROS accumulation, and enhanced MTX-induced cytotoxicity in vitro and in vivo. Collectively, our findings suggested that RSK2 activation could promote MTX resistance via upregulating SOX8 and attenuating MTX-induced ROS in GTN cells, which may help to develop experimental therapeutics to treat MTX-resistant GTN.Resistance to chemotherapy is frequently driven by aberrantly activated kinases. RSK2 was identified as a potential regulator of methotrexate resistance in gestational trophoblastic neoplastic cells. RSK2 activation promotes methotrexate resistance via upregulation of SOX8 and attenuation of reactive oxygen species. RSK2 inhibitors might therefore be useful to treat methotrexate-resistant gestational trophoblastic neoplasia.

Early prediction of post-molar gestational trophoblastic neoplasia and resistance to methotrexate, based on a single serum human chorionic gonadotropin measurement

BackgroundClinicians are unable to provide individualized counseling regarding risk of progression for patients with a complete hydatidiform mole (CHM). We developed nomograms enabling early prediction of post-molar gestational trophoblastic neoplasia (GTN) and resistance to methotrexate (MTX) based on a single serum human chorion gonadotropin (hCG) measurement. MethodsWe generated two nomograms with logistic regression: to predict post-molar GTN, and MTX resistance. For patients with high probability to progress to post-molar GTN or MTX resistance, we determined hCG cut-offs at 97.5% specificity to select patients for additional- or adjustments in current treatment. ResultsThe nomograms had a good to excellent ability to distinguish either between patients with uneventful hCG regression versus progression to post molar GTN, or between patients cured by MTX versus patients in whom resistance would occur. At 97.5% specificity, we identified 66% (95%CI 56–75) of the 149 patients who would progress to post-molar GTN, four weeks after initial curettage. For patients treated with MTX, we identified 55% (95%CI 23–83) of the 43 patients who would become resistant, preceding their third course at 97.5% specificity. ConclusionThe nomograms and cut-off levels can be used to assist in counseling for patients diagnosed with CHM.

NADPH Metabolism Determines the Leukemogenic Capacity and Drug Resistance of AML Cells

The mechanism by which redox metabolism regulates the fates of acute myeloid leukemia (AML) cells remains largely unknown. Using a highly sensitive, genetically-encoded fluorescent sensor of nicotinamide adenine dinucleotide phosphate (NADPH), iNap1, we found three heterogeneous subpopulations of AML cells with different NADPH levels in an MLL-AF9-induced murine AML model. The iNap1-high AML cells had enhanced proliferation capacities both in vitro and in vivo and were enriched for more functional leukemia-initiating cells (LICs) than their iNap1-low counterparts. Different NADPH levels in AML cells were also closely associated with their localization in the endosteal niche of bone marrow and chemotherapeutic resistance to methotrexate (MTX). Furthermore, iNap1-high human primary AML cells had enhanced proliferation abilities both in vitro and in vivo. Mechanistically, the MTHFD1-mediated folate cycle regulated NADPH homeostasis to promote leukemogenesis and MTX resistance. These results provide a new perspective for understanding mechanisms by which redox metabolism regulates cancer cell fate and provide a unique metabolic target for potential AML treatments.

Targeting serine hydroxymethyltransferases 1 and 2 for T-cell acute lymphoblastic leukemia therapy

Despite progress in the treatment of acute lymphoblastic leukemia (ALL), T-cell ALL (T-ALL) has limited treatment options, particularly in the setting of relapsed/refractory disease. Using an unbiased genome-scale CRISPR-Cas9 screen we sought to identify pathway dependencies for T-ALL which could be harnessed for therapy development. Disruption of the one-carbon folate, purine and pyrimidine pathways scored as the top metabolic pathways required for T-ALL proliferation. We used a recently developed inhibitor of SHMT1 and SHMT2, RZ-2994, to characterize the effect of inhibiting these enzymes of the one-carbon folate pathway in T-ALL and found that T-ALL cell lines were differentially sensitive to RZ-2994, with the drug inducing a S/G2 cell cycle arrest. The effects of SHMT1/2 inhibition were rescued by formate supplementation. Loss of both SHMT1 and SHMT2 was necessary for impaired growth and cell cycle arrest, with suppression of both SHMT1 and SHMT2 inhibiting leukemia progression in vivo. RZ-2994 also decreased leukemia burden in vivo and remained effective in the setting of methotrexate resistance in vitro. This study highlights the significance of the one-carbon folate pathway in T-ALL and supports further development of SHMT inhibitors for treatment of T-ALL and other cancers.

AHR-dependent genes and response to MTX therapy in rheumatoid arthritis patients

Methotrexate (MTX) is the first-line therapy for rheumatoid arthritis. Nevertheless, MTX resistance is quite a common issue in clinical practice. There are some premises that aryl hydrocarbon receptor (AhR) gene battery may take part in MTX metabolism. In the present retrospective study, we analyzed genes expression of AHR genes battery associated with MTX metabolism in whole blood of RA patients with good and poor response to MTX treatment. Additionally, sequencing, genotyping and bioinformatics analysis of AHR repressor gene (AHRR) c.565C > G (rs2292596) and c.1933G > C (rs34453673) have been performed. Theoretically, both changes may have an impact on H3K36me3 and H3K27me3. Evolutionary analysis revealed that rs2292596 may be possibly damaging. Allele G in rs2292596 and DAS28 seems to be associated with a higher risk of poor response to MTX treatment in RA. RA patients with poor response to MTX treatment revealed upregulated AhR and SLC19A1 mRNA level. Treatment with IL-6 inhibitor may be helpful to overcome the low-dose MTX resistance. Analysis of gene expression revealed possible another cause of poor response to MTX treatment which is different from that observed in the case of acute lymphoblastic leukemia.

ATRT-09. IDENTIFICATION OF POTENTIAL GENETIC DRIVERS OF METHOTREXATE (MTX) RESISTANCE IN ATYPICAL TERATOID RHABDOID TUMOURS (ATRT) THROUGH A GENOME-WIDE RNAI SCREEN

ATRT of the CNS constitute a group of rare and aggressive early childhood tumors with poor prognosis. While there are differing chemotherapeutic regimens for ATRT, high-dose MTX is a crucial component of many therapeutic protocols. Currently, the biological mechanisms contributing to the generation of MTX resistance in ATRT are unknown. To identify genes involved in MTX resistance in ATRT, an unbiased genome-wide RNAi screen on ATRT cell lines was conducted using 24,000 distinct shRNAs covering 8,000 genes. ATRT cells were transfected with a retrovirus containing pRS-shRNA vectors and treated with puromycin for selection. The resulting cells were treated with MTX to identify resistant clones and resistant colonies were then isolated and amplified individually. Presence of shRNA inserts in each colony was determined by PCR using pRS forward and reverse primers. PCR products within each of the three resistant colonies were sequenced, leading to the identification of three distinct genes, TGIF1, HIF3A and PGAM2, as potential indicators of resistance. Western blotting verified depletion of these proteins in their respective colonies. Proliferation assays were then conducted on cells from each resistant colony alongside control cells to confirm that the identified drivers conferred resistance. Sensitivity to MTX was significantly lower in TGIF1-depleted (IC50=212±8.48nM, n=3), HIF3A-depleted (IC50=52±4.68nM, n=3) and PGAM2-depleted (IC50=41±4.13nM, n=3) cells compared to control cells (IC50=19±2.87nM, n=3), (p<0.001). In addition, more than 60% of TGIF1, HIF3A, and PGAM2-depleted cells survived the maximum MTX treatment (100nM), while less than 20% of control cells survived this treatment. Our study using an unbiased genome-wide RNAi screen approach has shown that depletion of TGIF1, HIF3A and PGAM2 are potential molecular markers of MTX resistance in ATRT. Screening for their occurrence may help to identify patients at high risk of MTX resistance and may also serve as targets for future novel therapeutics development.

Doppler-based predictive model for methotrexate resistance in low-risk gestational trophoblastic neoplasia with myometrial invasion: prospective study of 147 patients.

ABSTRACTObjectivesThis prospective clinical study aimed to evaluate the vascularization characteristics of low‐risk gestational trophoblastic neoplasia (GTN) using Doppler imaging and to develop a predictive model for resistance to methotrexate (MTX).MethodsPatients with low‐risk GTN receiving primary MTX treatment were enrolled from the Women's Hospital, Zhejiang University School of Medicine, Hangzhou, China, from September 2012 to August 2018. The primary endpoint was to develop and internally validate a predictive model for resistance to MTX therapy in these patients. In the training set, clinical features and Doppler hemodynamic parameters before MTX therapy were analyzed using logistic regression to identify independent predictors of MTX resistance, which were integrated into the model. The predictive performance of the model was evaluated by leave‐one‐out cross‐validation in the training dataset and internal validation in an independent‐sample test dataset.ResultsThe entire imaging protocol was completed by 147 eligible patients, of which 110 comprised the training set and 37 the test set. In the training set, cases with myometrial invasion (81.8%; 90/110) showed vascular‐enriched areas in the myometrium and high velocity and low impedance ratios of the uterine artery (UtA) compared to cases without myometrial invasion (18.2%; 20/110). On multivariate logistic regression analysis, time‐averaged mean velocity in UtA (UtA‐TAmean) and the International Federation of Gynecology and Obstetrics (FIGO) score were identified as independent predictors (P = 0.009 and P = 0.043, respectively) of MTX resistance. The Doppler‐based predictive model, developed based on the 90 cases with myometrial invasion, was y = −2.95332 + 0.41696 × FIGO score + 0.03551 × UtA‐TAmean. The model showed an area under the curve of 0.757 (95% CI, 0.653–0.862) and the optimal cut‐off value was 0.50622, which had 45.2% sensitivity and 96.6% specificity. The model stratified patients with low‐risk GTN into low (< 10%), intermediate (10–90%) and high (> 90%) probability of MTX resistance, based on the threshold values of −1.59544 and 0.10046. The model had an accuracy of 74.4% (95% CI, 64.5–82.3%) in the cross‐validation and 72.7% (95% CI, 55.8–84.9%) in the internal validation.ConclusionsThe Doppler‐based predictive model, combining a non‐invasive marker of tumor vascularity with the FIGO scoring system, can differentiate cases with low from those with high probability of developing MTX resistance and therefore has the potential to guide treatment options in patients with low‐risk GTN and myometrial invasion. © 2020 Authors. Ultrasound in Obstetrics & Gynecology published by John Wiley & Sons Ltd on behalf of International Society of Ultrasound in Obstetrics and Gynecology.

Increasing the human chorionic gonadotrophin cut-off to ≤1000 IU/l for starting actinomycin D in post-molar gestational trophoblastic neoplasia developing resistance to methotrexate spares more women multi-agent chemotherapy

BackgroundA human chorionic gonadotropin (hCG) cut-off of ≤300 IU/l for starting actinomycin D (ActD) in post-molar gestational trophoblastic neoplasia (GTN) patients developing methotrexate resistance (MTX-R) reduced the number of women needing toxic multi-agent chemotherapy (etoposide, MTX and ActD alternating weekly with cyclophosphamide and vincristine; EMA/CO) without affecting survival. Here we assess whether an increased hCG cut-off of ≤1000 IU/l spares more women EMA/CO.Patients and methodsAll post-molar GTN patients treated with first-line methotrexate and folinic acid (MTX/FA) were identified in a national cohort between 2009 and 2016. Data collected included age, FIGO score, the hCG levels at MTX-R, and treatment outcomes.ResultsIn total, 609 GTN patients commenced treatment with MTX/FA achieving a complete response in 57% (348/609). Resistance developed in 25.1% (153/609) at an hCG ≤ 1000 IU/l and switching to ActD achieved remission in 92.8% without any major toxicity with the remaining 7.2% remitting on EMA/CO. Comparative analysis of patients switching at an hCG <100 versus 100-300 versus 300-1000 IU/l revealed a significant fall in the cure rate with second-line ActD from 97% (93/96) to 87% (34/39) to 78% (14/18), respectively, P = 0.009. However, by increasing the hCG cut-off from ≤300 to ≤1000 IU/l, 14 patients were spared EMA/CO chemotherapy. Moreover, in the present series, all post-molar GTN remain in remission.ConclusionThis study demonstrates that increasing the hCG cut-off from ≤300 to ≤1000 IU/l for choosing patients for ActD following MTX-R spares more women with GTN from the greater toxicity of EMA/CO without compromising 100% survival outcomes.

ABCB1 and SLCO1B1gene polymorphisms predict methotrexate-resistant for low-risk gestational trophoblastic neoplasia.

Aim: The aim of this study was to explore the effects of ABCB1 and SLCO1B1 gene polymorphisms and the methotrexate (MTX)treatment response in patients with low-risk gestational trophoblastic neoplasia (GTN). Materials & methods: Low-risk GTN patients who received MTX as a first-line single agent were enrolled. DNA was extracted from peripheral blood samples from 18 patients and assessed for ABCB1 C3435T and SLCO1B1 T521C. Results:ABCB1 C3435T and SLCO1B1 T521C polymorphisms were not associated with the MTX response or toxicity in Thai patients Conclusion: The selected ABCB1 and SLCO1B1 polymorphism do not predict the risk of MTX resistance in low-risk GTN.

Effect of mir-129 on the Sensitivity Enhancement of Methotrexate and Migration Inhibition in Osteosarcoma Cancer Cells

Background: MicroRNAs have been recently declared to be contributed to the various aspects of osteosarcoma cells, including growth and survival, apoptosis, invasion, and chemoresistance. Objectives: The present study aimed to investigate the potentiating effects of miR-129 on the chemosensitivity of Saose-2 osteosarcoma cells to methotrexate (MTX) and underlying mechanisms. Methods: Saose-2 cells were transfected with miR-129 mimics using Lipofectamine. The cytotoxic effects of miR-129 and MTX on Saose-2 cells were measured using MTT assay. Scratch wound healing assay was used to evaluate cell migration. The apoptosis rate of cancer cells was also measured using ELISA Cell Death Assay and flow cytometry. The mRNA expression levels of target genes were measured using quantitative RT-PCR. Results: miR-129 mimic transfection significantly increased the expression levels of this miRNA in Saose-2 cells (P&lt;0.05). The combination of MTX with miR-129 transfection led to enhanced cytotoxic effects of MTX in lower concentrations. In addition, miR-129 significantly increased MTX-induced apoptosis levels and decreased invasion behavior in Saose-2 cells. The mRNA expression levels of c-Myc, K-Ras, CXCR4, MMP9, and ADAMTS, as main genes involved in chemoresistance and invasion, were downregulated in miR-129 transfected cells. Conclusion: The obtained results revealed the importance of miR-129 in the sensitivity of osteosarcoma cells to MTX and its underlying mechanisms. Therefore, miR-129 might be an appropriate candidate for reversing MTX resistance in osteosarcoma cells.

Circular RNA circ_0081001 knockdown enhances methotrexate sensitivity in osteosarcoma cells by regulating miR-494-3p/TGM2 axis

BackgroundCircular RNAs (circRNAs) have been shown to participate in the chemoresistance and tumorigenesis of multiple cancers. The purpose of this research was to investigate the function of circ_0081001 in methotrexate (MTX) resistance of osteosarcoma (OS) and its potential molecular mechanism.MethodsThe expression of circ_0081001, cytochrome P450 family 51 subfamily A member 1 (CYP51A1), and miR-494-3p was detected by qRT-PCR. Cell viability, apoptosis, migration, and invasion were evaluated by Cell Counting Kit-8 (CCK-8) assay, flow cytometry, and transwell assay, respectively. Western blot (WB) assay was used to measure the protein levels of cleaved-caspase3 (cleaved-casp3), E-cadherin, N-cadherin, and transglutaminase-2 (TGM2). The interaction between miR-494-3p and circ_0081001 or TGM2 was predicted by bioinformatics analysis and verified using the dual-luciferase reporter assay. The mice xenograft model was established to investigate the roles of circ_0081001 in MTX resistance of OS in vivo.ResultsCirc_0081001 and TGM2 were upregulated, and miR-494-3p was downregulated in MTX-resistant OS tissues and cells. Moreover, circ_0081001 interference enhanced cell sensitivity to MTX through promoting apoptosis and inhibiting cell viability and metastasis in vitro. Furthermore, circ_0081001 was identified as a molecular sponge of miR-494-3p to upregulate TGM2 level. In addition, circ_0081001 knockdown inhibited MTX resistance via upregulating miR-494-3p and downregulating TGM2. Besides, circ_0081001 downregulation improved MTX sensitivity of OS in vivo.ConclusionKnockdown of circ_0081001 enhanced MTX sensitivity of OS cells through downregulating TGM2 by sponging miR-494-3p, elucidating a novel regulatory mechanism for chemoresistance of OS and providing a potential circRNA-targeted therapy for OS.

SLAMF1 Promotes Methotrexate Resistance via Activating Autophagy in Choriocarcinoma Cells.

ObjectiveThe acquisition of chemoresistance to methotrexate (MTX) still remains one of the major challenges for choriocarcinoma treatment. Herein, we aimed to evaluate the potential role of Signaling Lymphocytic Activation Molecule Family Member 1 (SLAMF1) as a possible regulator of chemoresistance to MTX in choriocarcinoma.Material and MethodsMTX-resistant JEG3 and JAR sublines (JEG3/MTX, JAR/MTX) were used to study SLAMF1 function. CCK8 assay and soft agar assay were conducted to measure the cell viability and clonogenesis of choriocarcinoma cells, respectively; MDC incorporation assay was conducted for the quantification of intracellular autophagy; BrdU labeling was used to assess the proliferative potential of choriocarcinoma cells; SLAMF1 protein expression was analyzed by Western blotting.ResultsUpregulation of SLAMF1 expression was observed in MTX-resistant JEG3/MTX and JAR/MTX sublines compared to their parental JEG3 and JAR cell lines, respectively. Knockdown of SLAMF1 markedly attenuated cell viability and soft agar clonogenesis after incubation with MTX in JEG3/MTX and JAR/MTX cells. In contrast, constitutive expression of SLAMF1 rescued cell survival soft agar clonogenesis in JEG3 and JAR cells treated with MTX. Moreover, autophagy is apparently activated in MTX-resistant JEG3/MTX and JAR/MTX sublines compared to their parental cell lines. Autophagy inhibitor 3-methyladenine and bafilomycin A1 enhanced MTX-induced cytotoxicity in MTX-resistant JEG3 and JAR sublines. Further, SLAMF1 might activate autophagy-related mechanism to promote resistance to MTX in choriocarcinoma cells. Depletion of SLAMF1 suppressed autophagy and induced apoptosis in MTX-treated JEG3/MTX and JAR/MTX cells.ConclusionSLAMF1 might promote MTX resistance via activating protective autophagy in choriocarcinoma cell lines. Targeting SLAMF1 might be a useful therapeutic strategy to sensitize choriocarcinoma cells to MTX-based regimens.

Sonographic characteristics of post-molar gestational trophoblastic neoplasia at diagnosis and during follow-up, and relationship with methotrexate resistance.

To describe the sonographic characteristics of post-molar gestational trophoblastic neoplasia (GTN) at diagnosis and during follow-up, and to assess their association with methotrexate (MTX) resistance (R) as first-line chemotherapy. This was a retrospective study of all women treated for post-molar GTN at Karolinska University Hospital, Stockholm, Sweden, between October 2010 and December 2017, who had undergone expert transvaginal ultrasound assessment ≤ 2 weeks prior to, or ≤ 1 week after, the start of first-line MTX treatment. Women with a detectable uterine lesion were followed up with repeat scans during treatment, as well as after treatment in cases of persistent lesions. The association between MTX-R and sonographic findings at inclusion was assessed. Of 47 eligible women, 36 were included in the analysis after excluding those who had not undergone structured transvaginal ultrasound assessment and those who started treatment at another center. The median age at diagnosis was 33 (interquartile range (IQR), 27-43) years and 35/36 (97%) women were in the FIGO low-risk group (risk score, 0-6). At inclusion, no uterine lesions were found in eight (22%) women, focal lesions in 24 (67%) women and global lesions in four (11%) women. Median maximum lesion diameter was 40.4 (IQR, 31.3-49.4) mm and 26/28 (93%) lesions had a color score of 3 or 4. Arteriovenous fistulas were found in 9/28 (32%) women and theca lutein cysts in 4/36 (11%) women. Four women with GTN lesion at inclusion underwent hysterectomy prior to the first follow-up ultrasound scan and a fifth woman with a growing lesion underwent hysterectomy, which revealed persistent viable trophoblastic tissue. All remaining women reached complete remission and median time to human chorionic gonadotropin normalization was 2.7 (IQR, 1.4-3.7) months. During ultrasound follow-up, 88% (21/24) of lesions resolved completely. Two women with a persisting lesion remained in complete remission. Median time to disappearance of vascularity was 5.8 (IQR, 3.7-9.3) months and median time to resolution of the lesion was 6.8 (IQR, 3.7-9.3) months. MTX-R developed in 12/31 (39%) women. Uterine tumors ≥ 4 cm (73% vs 17%; P = 0.008) and global lesions (25% vs 0%; P = 0.03) were significantly more common in women with compared to those without MTX-R. Uterine lesions were detected at the time of diagnosis in 78% of women with post-molar GTN. The vast majority of the lesions resolved completely during follow-up, after a median of 7 months. MTX-R was more common in uterine tumors of 4 cm, or larger, and in global lesions. Copyright © 2020 ISUOG. Published by John Wiley & Sons Ltd.

Prognosis of Gestational Trophoblastic Neoplasia in Women at 40 Years Old and Above: A Multicentre Retrospective Study

Purpose: To investigate the outcome of different treatment strategies in patients with gestational trophoblastic neoplasia (GTN) in women at 40 years old and above. Patients and Methods: We analysed a historical cohort from 5 referral centres from 5 countries, including all women with GTN treated between 2012 and 2017, who were 40 years old and older. Baseline characteristics and outcome of different treatment strategies were recorded and evaluated. The patients were categorized into low-risk non-metastatic, low-risk metastatic and high-risk, based on the FIGO classification. Results: A total of 141 cases were identified, of which 112 cases fulfilled the inclusion criteria. Mean age was 45.4 years ± 4.2SD. Of 80 patients with LR non-metastatic GTN, 46 women received single agent chemotherapy and 34 a hysterectomy with or without (n = 4) chemotherapy. Higher remission rate and shorter treatment duration (P=0.001) was seen in the group that underwent hysterectomy. Seven of the 14 patients with low-risk, metastatic GTN were cured with methotrexate. Two of the 18 high risk patients died before treatment, four were treated with polychemotherapy; two of them needed second line chemotherapy for incomplete response. Two cases received induction with methotrexate followed by EMA/CO. Ten highrisk patients were treated with hysterectomy and chemotherapy, of these six achieved complete remission, three needed second line chemotherapy, and one patient died during chemotherapy treatment. Conclusion: In this cohort of women with GTN at 40 years old or above, we found high proportions of metastatic and high-risk cases, of methotrexate resistance, and of need for multiple treatment lines. In all groups, hysterectomy was performed, but its role remains controversial in metastatic low-risk and high-risk disease.

HSH2D contributes to methotrexate resistance inhuman T‑cell acute lymphoblastic leukaemia.

Acute lymphoblastic leukaemia (ALL) is a malignant proliferative disease that originates from B-lineage or T-lineage lymphoid progenitor cells. Resistance to chemotherapy remains an important factor for treatment failure. The aim of the present study was to investigate drug resistance in T-cell ALL (T-ALL). Bioinformatics analysis of Oncomine and Gene Expression Omnibus data was performed to evaluate the expression of haematopoietic SH2 domain containing (HSH2D) in various lymphomas. HuT-78 cells with HSH2D overexpression and or knockdown were constructed, and the effect on related downstream signalling molecules was detected. To study the effect of HSH2D on methotrexate (MTX) resistance, cell cycle and apoptosis analyses were conducted using flow cytometry, and MTT and EdU assays were used to detect the effect of MTX resistance and HSH2D gene expression on the biological function of HuT-78 cells. Via the analysis of the data sets, it was identified that the expression of HSH2D was downregulated in T-ALL compared with B-cell ALL. Western blotting and reverse transcription-quantitative PCR demonstrated that the overexpression of HSH2 resulted in the inhibition of CD28-mediated IL-2 activation. In related experiments with drug-resistant cell lines, it was determined that HSH2D expression is necessary for HuT-78 cells to be resistant to MTX. In conclusion, the results suggested that HSH2D serves an important role in the resistance of T-ALL to MTX, which provides a potential research target for the study of drug resistance of T-ALL.

Association of relapse-linked ARID5B single nucleotide polymorphisms with drug resistance in B-cell precursor acute lymphoblastic leukemia cell lines

BackgroundThe genetic variants of the ARID5B gene have recently been reported to be associated with disease susceptibility and treatment outcome in childhood acute lymphoblastic leukemia (ALL). However, few studies have explored the association of ARID5B with sensitivities to chemotherapeutic agents.MethodsWe genotyped susceptibility-linked rs7923074 and rs10821936 as well as relapse-linked rs4948488, rs2893881, and rs6479778 of ARDI5B by direct sequencing of polymerase chain reaction (PCR) products in 72 B-cell precursor-ALL (BCP-ALL) cell lines established from Japanese patients. We also quantified their ARID5B expression levels by real-time reverse transcription PCR, and determined their 50% inhibitory concentration (IC50) values by alamarBlue assays in nine representative chemotherapeutic agents used for ALL treatment.ResultsNo significant associations were observed in genotypes of the susceptibility-linked single nucleotide polymorphisms (SNPs) and the relapsed-linked SNPs with ARID5B gene expression levels. Of note, IC50 values of vincristine (VCR) (median IC50: 39.6 ng/ml) in 12 cell lines with homozygous genotype of risk allele (C) in the relapse-linked rs4948488 were significantly higher (p = 0.031 in Mann–Whitney U test) than those (1.04 ng/ml) in 60 cell lines with heterozygous or homozygous genotypes of the non-risk allele (T). Furthermore, the IC50 values of mafosfamide [Maf; active metabolite of cyclophosphamide (CY)] and cytarabine (AraC) tended to be associated with the genotype of rs4948488. Similar associations were observed in genotypes of the relapse-linked rs2893881 and rs6479778, but not in those of the susceptibility-linked rs7923074 and rs10821936. In addition, the IC50 values of methotrexate (MTX) were significantly higher (p = 0.023) in 36 cell lines with lower ARID5B gene expression (median IC50: 37.1 ng/ml) than those in the other 36 cell lines with higher expression (16.9 ng/ml).ConclusionThese observations in 72 BCP-ALL cell lines suggested that the risk allele of the relapse-linked SNPs of ARID5B may be involved in a higher relapse rate because of resistance to chemotherapeutic agents such as VCR, CY, and AraC. In addition, lower ARID5B gene expression may be associated with MTX resistance.

The advances of methotrexate resistance in rheumatoid arthritis

Rheumatoid arthritis (RA) is a systemic autoimmune disease, which is characterized by a chronic fluctuating course and immune dysfunction, resulting in affecting the health and life quality of RA patients. Methotrexate (MTX), as the standard gold treatment of RA, has received more and more clinical applications and basic pharmacological research. In several observational studies, MTXR, and treatment responses in RA patients show that the ratio of MTXR and non- response is about 30%-50%, namely MTX resistance (MTXR). Extensive efforts have been made into the investigation of the mechanism and effective biomarkers in MTXR of RA. In this paper, we discuss the recent findings regarding the critical signaling pathways of MTXR in RA. Provide research targets and directions for a drug therapy that develop preventive strategies and effective treatments of MTXR.

Uterine artery pulsatility index and serum BMP-9 predict resistance to methotrexate therapy in gestational trophoblastic neoplasia: A cohort study

Methotrexate is the most common first-line chemotherapy for low-risk gestational trophoblastic neoplasia (GTN). Uterine artery pulsatility index (UAPI) is an ultrasound marker for tumor vascularity that has been associated with an increased risk of methotrexate resistance. The combination of circulating angiogenic factor levels with UAPI data may improve the capacity of this model to predict chemoresistance. This was a single-center cohort study of women newly diagnosed between January 2008 and June 2012 with low-risk GTN during postmolar surveillance and treated with single-agent methotrexate at Charing Cross Hospital, a UK national center for treatment of gestational trophoblastic disease. Two hundred seventeen women underwent an ultrasound for UAPI measurement prior to initiation of chemotherapy. To examine serologic markers of methotrexate resistance among this cohort, we performed a case-control study using archived serum from 76 patients who could be matched based on prognostic risk score. Serum samples were examined by immunoassay to measure 8 different angiogenic factors (VEGF-A, FGF-basic, PLGF-1, PDGF-BB, EGF, ANGPT2, BMP-9, and ENG). Receiver-operator characteristic area under the curve (AUC) values were calculated for the ability of each analyte to correctly classify patients as methotrexate sensitive (MTX-S) or resistant (MTX-R). Total human chorionic gonadotropin levels were similar between the MTX-S and MTX-R groups. UAPI values were significantly higher in MTX-S (median 1.30 [interquartile range {IQR} = 0.80-1.90]) compared to MTX-R patients (median 0.875 [IQR = 0.60-1.30]; P < 0.0001) with AUC 0.68 (95% confidence interval 0.61-0.76; P < 0.0001). In univariate analysis, only BMP-9 concentrations were significantly different between groups, lower among MTX-S (median of 225 ng/L, IQR = 170-287) compared to MTX-R patients (median 280 ng/L [IQR = 200-339]; P= 0.03). Combining UAPI with BMP-9 concentration improved prediction for chemoresistance with AUC 0.77 (95% confidence interval 0.66-0.88; P < 0.0001). Circulating levels of BMP-9 are elevated in newly diagnosed women with low-risk GTN destined to fail primary methotrexate therapy. A combined test using serum BMP-9 plus UAPI might improve prediction of MTX-R in low-risk GTN.

Circular RNA hsa_circ_0000073 contributes to osteosarcoma cell proliferation, migration, invasion and methotrexate resistance by sponging miR-145-5p and miR-151-3p and upregulating NRAS.

An increasing number of studies have demonstrated that circular RNAs (circRNAs), as promising therapeutic targets, are essential for diverse human diseases, especially cancer. However, the potential functions and complex mechanisms of most circRNAs in osteosarcoma (OS) are still not fully elucidated. In the present study, we obtained the expression profile of circRNAs from a GEO database (GSE96964) and identified hsa_circ_0000073 as a highly expressed candidate in OS. Overexpression of hsa_circ_0000073 accelerated the proliferation, migration, invasion and MTX resistance of OS cells, and knockdown of hsa_circ_0000073 resulted in the opposite effects. Mechanistically, hsa_circ_0000073 upregulated NRAS expression by targeting miR-145-5p and miR-151-3p in OS cells. In addition, the promotion of OS progression by hsa_circ_000007 was blocked by miR-145-5p and miR-151-3p-mediated NRAS inhibition. In conclusion, hsa_circ_0000073 facilitated the proliferation, migration, invasion and MTX resistance of OS cells through the inhibition of miR-145-5p and miR-151-3p-mediated downregulation of NRAS.

MTHFR C677T and TS 5’-UTR 3R/3R Gene Polymorphism in Methotrexate-Resistant Childhood Acute Lymphoblastic Leukemia

BACKGROUND: Childhood acute lymphoblastic leukemia (ALL) is the most common pediatric malignancy in Indonesia and often treated by methotrexate (MTX). Though it can be cured in 30-60% of patients, MTX resistance remains the major cause of treatment failure in childhood ALL. Previous sudies showed that its anti-leukemic property was moderated by MTX ability to inhibitmethylene tetra hydrofolate reductase (MTHFR) and thymidylate synthase (TS) in folate metabolism. This study investigates the correlation between MTHFR and TS polymorphism and MTX resistance in ALL children.METHODS: A total of 155 subjects obtained from all subjects prior to chemotherapy. DNA from blood samples were extracted and underwent polymerase chain reactionrestriction fragment length polymorphism (PCR-RFLP) to evaluate MTHFR C677T and TS 5’-UTR 3R/3R polymorphism.RESULTS: There was significant correlation between MTHFR C677T and TS 5’-UTR 3R/3R gene polymorphism with MTX resistance. Subjectswith MTHFR C677T and TS 5’-UTR 3R/3R gene polymorphism were 4 times (p=0.007) and 6.4 times (p=0.001) more likely to be MTX resistant than those without gene polymorphisms, respectively.CONCLUSION: MTHFR C677T andTS 5’-UTR 3R/3R represent dominant gene polymorphism related to MTX resistance in childhood ALL.KEYWORDS: gene polymorphism, folate metabolism, acute lymphoblastic leukemia