Optimization of lens size and ion source parameters for gas chromatography/mass spectrometry-based metabolomics using hydrogen as a carrier gas.

Introduction: Isocitrate dehydrogenase 1 (IDH1) is mutated in various types of human cancers, predominantly an arginine to histidine amino acid change at codon 132. This structural alteration drives the catalysis of α-ketoglutarate to the oncometabolite D-2-hydroxyglutarate (2HG), which enhances DNA and protein hypermethylation and cellular dedifferentiation. Pharmacologic inhibitors of the mutant IDH1 protein show promise in clinical trials, yet the regulation of IDH1 has not been fully elucidated. We recently discovered in wild type IDH1 tumors that the regulatory RNA-binding and stress response protein, HuR (ELAVL1), protects cancer cells under nutrient deprivation by regulating the expression of core metabolic enzymes, including IDH1. We mapped the regulatory HuR binding site on the IDH1 transcript to the 3’-untranslated region. Since wild type and mutant IDH1 alleles both contain this binding sequence, we hypothesize that HuR is an important regulator of both isozymes and is biologically important in mutant IDH1 tumors. Methods: HuR expression was suppressed by siRNA in a fibrosarcoma cell line (HT-1080) harboring a natural heterozygous IDH1 mutation and cell viability was determined by PicoGreen and Trypan blue exclusion assays. Sensitivity to pharmacologic inhibition of the mutant IDH1 protein was assessed. Sanger sequencing and mRNP-IP were performed to determine HuR's impact on the expression of each IDH1 allele. To further characterize the post-transcriptional regulation of IDH1 by HuR, CRISPR/CAS 9 editing of the HuR gene was performed. Results: Sanger sequencing of IDH1 after depletion of HuR in HT1080 cells, as well as after HuR mRNP-IP, revealed that HuR regulates both the mutant and wild type IDH1. Drug sensitivity assays to a mutant IDH1 inhibitor (AGI-5198) under varying glucose concentrations revealed glucose deprivation to be a novel driver of chemo-resistance. However, HuR silencing abrogated HT1080 resistance to AGI-5198 under these conditions. Targeted knockout of HuR using the CRISPR/CAS9 system resulted in potent suppression of mutant and wild type IDH1, at both the mRNA and protein levels when compared to CRISPR/Cas9 control. Conclusions: These findings reveal that harsh metabolic conditions present in the tumor microenvironment induce chemo-resistance in an IDH1 mutant cell line to a mutant IDH1 inhibitor, in an HuR dependent manner. Moreover, both mutant and wild type IDH1 alleles are potently regulated by HuR. Therapeutic strategies that target the HuR-IDH1 axis and block this resistance mechanism may enhance the efficacy of mutant IDH1 inhibitors for relevant tumors. Citation Format: Mahsa Zarei, Shruti Lal, Nicole C. Mambelli-Lisboa, Edwin Cheung, Saswati N. Chand, Charles J. Yeo, Jonathan R. Brody, Jordan M. Winter. The mRNA-binding protein HuR, regulates mutant and wild type IDH1 expression in IDH1-mutated cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1847.

Mutational Analysis for IDH1 and IDH2 In Pediatric Leukemia

Objective To establish a human colon cancer cell line HCT-116/5-FU resistant to 5-fluorouracil(5-FU)and explore the relationship between runt-related transcription factor 3(RUNX3)and drug resistance of colorectal cancer.Methods The human colon cancer cell line HCT-116/5-FU with resistance to 5-FU was established by low concentration gradient increment combined with high-dose intermittent shock.CCK-8 method was used to determine the half maximal inhibitory concentration(IC50)of 5-FU on the parent line HCT-116 and drug-resistant line HCT-116/5-FU.The cell growth curve was established for the calculation of population doubling time(TD).The mRNA levels and protein levels of RUNX3,P-glycoprotein(P-gp),multidrug resistance-associated protein 1(MRP1),and lung resistance-related protein(LRP)in HCT-116 and HCT-116/5-FU cells were determined by qRT-PCR and Western blotting,respectively.The RUNX3 expression in HCT-116 cells was knocked down by siRNA technique,and the cells were divided into RUNX3 knockdown groups(si-RUNX3-1 group and si-RUNX3-2 group)and negative control group(si-NC group).The knockdown efficiency was verified by qRT-PCR at the mRNA level and Western blotting at the protein level.The IC50 in si-RUNX3 groups and si-NC group was determined with CCK-8 method,and the expression of P-gp,MRP1,and LRP in the two groups was detected by Western blotting.Results A stable human colon cancer drug-resistant cell line HCT-116/5-FU was successfully constructed.HCT-116/5-FU showed the TD 1.38 times as long as that of HCT-116(P=0.002)and changed morphology.The mRNA level of RUNX3 in HCT-116/5-FU cells was significantly lower than that in HCT-116 cells(P=0.048),and those of P-gp(P=0.008),MRP1(P=0.001),and LRP(P=0.001)showed the opposite trend.The protein level of RUNX3 in HCT-116/5-FU cells was significantly lower than that in HCT-116(P<0.001),and those of P-gp,MRP1,and LRP presented the opposite trend(all P<0.001).The HCT-116 cell model with low expression of RUNX3 was successfully established.The mRNA level of RUNX3 had no significant difference between si-RUNX3-1 group and si-NC group(P=0.064),while the level in si-RUNX3-2 group was significantly lower than that in si-NC group(P=0.034).The protein levels of RUNX3 in si-RUNX3-1 group and si-RUNX3-2 group were lower than that in si-NC group(both P<0.001).The results demonstrated higher knocking efficiency in si-RUNX3-2 group,which was thus selected to complete the follow-up test.The IC50 of si-RUNX3 group was significantly higher than that of si-NC group(P<0.001),which indicated that the down-regulated expression of RUNX3 could reduce the sensitivity of HCT-116 cells to 5-FU.The relative protein levels of P-gp,MRP1,and LRP in si-RUNX3 group were significantly higher than those in si-NC group(all P<0.001).Conclusion The down-regulation of RUNX3 expression can reduce the sensitivity of HCT-116 cells to 5-FU,which is considered to be related to the up-regulated expression of P-gp,MRP1,and LRP.

Identification of a CpG Island Methylator Phenotype that Defines a Distinct Subgroup of Glioma

Colorectal cancer (CRC) remains a significant global health challenge, driving increased interest in the potential preventive effects of dietary bioactive components. Ganoderma lucidum spore oil (GSO) is a food-grade lipid extract rich in bioactive fatty acids and triterpenoid compounds, yet its mechanism of action against CRC from a food science perspective remains incompletely elucidated. This study employed an integrated approach combining gas chromatography-mass spectrometry (GC-MS) analysis, network pharmacology prediction, and in vitro validation to preliminarily elucidate the mechanism by which GSO inhibits CRC proliferation. GC-MS identified key lipophilic constituents, while network pharmacology analysis revealed the PPAR signalling pathway and lipid metabolism as central to GSO's potential effects. In vitro experiments confirmed that GSO significantly inhibited the proliferation of the human colon cancer cell line HCT116 in a dose-dependent manner. Mechanistically, GSO treatment simultaneously upregulated PPAR subtype expression (PPARA, PPARD, PPARG) at both the protein and mRNA levels, while downregulating key lipid transport (FABP1, FABP4) and inflammatory (PTGS1) mediators. Molecular docking further confirmed the high-affinity interactions between GSO's primary constituents (linoleic acid, oleic acid, ganoderic acid C) and these core targets. the findings suggest that GSO as a source of bioactive lipids may exert anti-colorectal cancer effects primarily through regulating PPAR signalling pathways and associated lipid metabolism. These findings indicated that as a source of bioactive lipids, GSO may exert its anti-colorectal cancer effects primarily through regulating the PPAR signalling pathway and associated lipid metabolism. This study provides scientific evidence supporting the consideration of GSO as a potential functional food ingredient for colorectal cancer prevention and dietary intervention.

BackgroundMutations in isocitrate dehydrogenase 1 (IDH1) and isocitrate dehydrogenase 2 (IDH2) are frequent in low-grade gliomas and secondary glioblastomas (sGBM). Because they yield the same oncometabolite, D-2-hydroxyglutarate, they are often treated as equivalent and pooled. The objective of this study was to provide insight into the differences between IDH1 and IDH2 mutant gliomas.MethodsTo investigate the different clinical and molecular characterization between IDH1 mutant and IDH2 mutant gliomas, we studied 811 patients with IDH1 mutations, IDH2 mutations and IDH1/2 wild-type. In addition, whole-transcriptome sequencing and DNA methylation data were used to assess the distribution of genetic changes in IDH1 and IDH2 mutant gliomas in a Chinese population-based cohort.ResultsAmong 811 gliomas in our cohort, 448 cases (55.2 %) harbored an IDH1 mutation, 18 cases (2.2 %) harbored an IDH2 mutation and 345 cases (42.6 %) harbored an IDH1/2 wild-type. We found that IDH1 and IDH2 are mutually exclusive in gliomas, and IDH2 mutations are mutually exclusive with PTEN, P53 and ATRX mutations. Patients with IDH2 mutations had a higher frequency of 1p/19q co-deletion (p < 0.05) than IDH1 mutant patients. In addition, a Gene Set Enrichment Analysis (GSEA) showed that IDH2 mutant gliomas were associated with the oxidative phosphorylation gene set, and the four most representative biological processes for genes commonly altered by hypermethylation in IDH2 mutant gliomas were the regulation of cell proliferation, cell motion, cell migration and response to hypoxia. Patients with IDH2 mutant gliomas exhibited longer Overall survival (OS) (p < 0.05) and longer Progression-free survival (PFS) (p < 0.05) than patients with IDH1/2 wild-type gliomas. However, their OS and PFS did not differ from that of IDH1 mutant patients.ConclusionsOur study revealed an intrinsic distinction between IDH1 and IDH2 mutant gliomas, and these mutations should be considered separately because their differences could have implications for the diagnosis and treatment of IDH1/2 mutant gliomas.

To investigate the effects on telomerase activity of transfection of human T-STAR gene full-length sense cDNA or partial antisense cDNA into human colon cancer cell line HCT-116. mRNA and protein expression levels of T-STAR gene were determined by RT-PCR and western blot, and telomerase activity was measured by PCR-ELISA, after transfection of T-STAR sense or antisense gene into HCT-116 cells with lipofectamine. T-STAR gene expression was enhanced or knocked down both at mRNA and protein levels, and telomerase activity was significantly increased or decreased. The T-STAR gene may participate in regulation of telomerase activity in human colon cancer HCT-116 cells in a parallel fashion.

Non-coding RNAs (ncRNAs) are finely tuned cellular regulators important for human cell growth and cancer progression. DUBR (Dppa2 upstream binding RNA, also known as linc00883) is a nuclear ncRNA first discovered in mice for its role in regulating myoblast differentiation through interactions with chromatin and DNA methyltransferases. High expression levels of this ncRNA are predictive of poor patient outcome in colon adenocarcinoma, suggesting that DUBR may be involved in controlling cancer growth. To elucidate its function, we used RAP-MS and RNA immunoprecipitation techniques which revealed its interaction with epigenetic maintenance proteins in the human colon cancer cell line HCT116. Further, ATAC-seq and RNA-seq were used to address its function in regulating the epigenome and transcriptome of HCT116 cells. Here we report that DUBR is a regulator of human colon cancer cell line HCT116 survival. Additionally, we find that the ncRNA DUBR regulates AP-1 transcription factor binding site accessibility at enhancers of genes involved in differentiation and morphogenesis through interactions with epigenetic proteins such as NuRD complex members HDAC1 and CHD4.

Anti-cancer potential of polysaccharide extracted from hawthorn (Crataegus.) on human colon cancer cell line HCT116 via cell cycle arrest and apoptosis

AIMTo investigate the role of calmodulin-dependent protein kinase II (CaMKII) in colon cancer growth, migration and invasion.METHODSCaMKII expression in colon cancer and paracancerous tissues was evaluated via immunochemistry. Transcriptional and posttranscriptional levels of CaMKIIin tissue samples and MMP2, MMP9 and TIMP-1 expression in the human colon cancer cell line HCT116 were assessed by qRT-PCR and western blot. Cell proliferation was detected with the MTT assay. Cancer cell migration and invasion were investigated with the Transwell culture system and wound-healing assay.RESULTSWe first demonstrated that CaMKII was over-expressed in human colon cancers and was associated with cancer differentiation. In the human colon cancer cell line HCT116, the CaMKII-specific inhibitor KN93, but not its inactive analogue KN92, decreased cancer cell proliferation. Furthermore, KN93 also significantly prohibited HCT116 cell migration and invasion. The specific inhibition of ERK1/2 or p38 decreased the proliferation and migration of colon cancer cells.CONCLUSIONOur findings highlight CaMKII as a potential critical mediator in human colon tumor development and metastasis.

Several essential oils possess pharmacological effects. Among the various constituents of essential oils, 1, 8-cineole has been shown to possess pharmacological effects such as anti-bacterial and anti-inflammatory effects. The effect of 1, 8-cineole on human colorectal cancer cells, however, has not reported previously. In this study, we have investigated the anti-proliferative effect of 1, 8-cineole on human colon cancer cell lines HCT116 and RKO by WST-8 and BrdU assays. The cytotoxicity of 1, 8-cineole was investigated by LDH activity and TUNEL staining. The mechanism of apoptosis by 1, 8-cineole was determined by western blot analyses. In in vivo study, RKO cells were injected into the SCID mice and the effect of 1, 8-cineole was investigated. Specific induction of apoptosis, not necrosis, was observed in human colon cancer cell lines HCT116 and RKO by 1, 8-cineole. The treatment with 1, 8-cineole was associated with inactivation of survivin and Akt and activation of p38. These molecules induced cleaved PARP and caspase-3, finally causing apoptosis. In xenotransplanted SCID mice, the 1, 8-cineole group showed significantly inhibited tumor progression compared to the control group. These results indicated 1, 8-cineole suppressed human colorectal cancer proliferation by inducing apoptosis. Based on these studies 1, 8-cineole would be an effective strategy to treat colorectal cancer.

Objective To investigate the relationship between isocitrate dehydrogenase 1 (IDH1)gene mutation and the pathological grade and prognosis in astrocytic glioma patients.And to identify whether DNA methylation is one of the IDH1 gene regulatory patterns.Methods IDH1 mutation was detected by direct sequencing in 91 astrocytic glioma tissue samples; DNA methylation of ID-H1 promoter was detected by bisulfite sequencing in 9 astrocytic glioma cases.Results IDH1 mutations were demonstrated in 35 of 91 cases (38.5％) and all mutations were the type of R132H.No mutation of IDH1 was detected in WHO grade Ⅰ astrocytoma.Frequent mutations of IDH1 were observed as 70.6％ in primary WHO grade Ⅱ astrocytoma,62.1％ in primary WHO grade Ⅲ astrocytoma,12.5％ in primary glioblastoma multiforme.Frequencies of IDH1 mutations were negatively correlated with WHO pathological grade Ⅱ ～ Ⅳ of primary astrocytic glioma (r =0.522,P ＜0.001).IDH1 mutations were found in 5 of 7 secondary glioblastomas (71.4％),and the type of mutation was consistent with that in primary tumors.The mutation of the IDH1 was significantly associated with better outcome in the patients of astrocytic gliomas (P ＜ 0.05).DNA methylation of IDH1 promoter was not found in 9 glioma patients.Conclusions IDH1 mutation occured frequently in astrocytic gliomas and is a favorable prognostic indicator.DNA methylation might not be involved in the regulation of IDH1 gene expression. Key words: Glioma; IDH1 ; Mutation; DNA methylation

IDH1/2 gene hotspot mutations in central nervous system tumours: analysis of 922 Chinese patients

Isocitrate dehydrogenase-1 mutations: a fundamentally new understanding of diffuse glioma?

2082 Background: In gliomas, relationship between radiological characteristics and several biomarkers was the subject of numerous publications. Mutations in the isocitrate dehydrogenase 1 (IDH1) gene have been identified recently to play a key role in these tumors occuring in up to 75% of low-grade diffuse (WHO grade II) and anaplastic (WHO grade III) astrocytic, oligodendroglial and mixed oligodendroglial neoplasms. However, the correlation with magnetic resonance imaging (MRI) features has been little studied. Methods: Patients treated for WHO grade II and III oligodendroglial tumors between 2005 and 2011 were retrospectively identified. Each case has been reviewed by the same neuropathologist. IDH1 and IDH2 mutations were available. Preoperative MRI, including T1 weighted, T2 weighted, T1 contrast enhanced, FLAIR, T2* weighted, diffusion weighted (ADC ratio), perfusion weighted (CBV ratio) and MR spectroscopy, were analyzed by two radiologists blinded from molecular data. Logistic regression analysis and Fisher’s test were used to develop predictive models of genetic profile from imaging. Results: Sixty eight patients, WHO grade II (n= 37) and grade III (n=31) patients were identified. Mean age at diagnosis was 46 years; ratio male/female was 40/28. IDH1 mutations were identified in 42 patients (62 %), IDH2 in 4 patients (6 %). Analysis of tumor location, size, borders, morphological aspect, and signal did not shown any significant difference between IDH1 mutated group and IDH1 non mutated group neither in grade II nor in grade III oligodendroglial tumors. In the same way, MR spectroscopy (Choline/NAA ratio and detection of lipid and lactate) was not relevant to discern the two groups. As well, ADC ratio (1,5 versus 1,4; p=0,35) and CBV ratio (3,4 versus 4,2; p= 0,46) did not reveal any difference between mutated group and non mutated group. Conclusions: In our study, IDH1 mutations were not correlated with MRI features available during routine MRI. Nevertheless, recent studies suggest the ability of MR spectroscopy to detect 2-hydroxyglutarate as an MRI marker of IDH1 mutated tumors, which encourage carrying on research in molecular imaging.