Selenoprotein P1 as a biomarker of insulin resistance in pediatric obesity:Insights and implications

This editorial discusses the findings of Elbarky et al on the role of selenoprotein P1(SEPP1)in pediatric obesity and insulin resistance.Their study uncovered si-gnificantly lower SEPP1 Levels in children who were obese compared with hea-lthy peers,demonstrating a negative correlation between SEPP1 levels and mea-sures of adiposity and insulin resistance.These findings suggest that SEPP1 is a biomarker useful in the early identification of insulin resistance in pediatric populations.This editorial emphasizes the clinical implications of the study and calls for further research to validate and explore the role of SEPP1 in metabolic health.

Understanding the link between type 2 diabetes mellitus and Parkinson's disease:role of brain insulin resistance

Type 2 diabetes mellitus and Parkinson's disease are chronic diseases linked to a growing pandemic that affects older adults and causes significant socio-economic burden.Epidemiological data supporting a close relationship between these two aging-related diseases have resulted in the investigation of shared pathophysiological molecular mechanisms.Impaired insulin signaling in the brain has gained increasing attention during the last decade and has been suggested to contribute to the development of Parkinson's disease through the dysregulation of several pathological processes.The contribution of type 2 diabetes mellitus and insulin resistance in neurodegeneration in Parkinson's disease,with emphasis on brain insulin resistance,is extensively discussed in this article and new therapeutic strategies targeting this pathological link are presented and reviewed.

Molecular Evolution of Rice Blast Resistance Gene bsr-d1

Rice blast,caused by the fungus Magnaporthe oryzae,reduces rice yields by 10%to 35%.Incorporating blast resistance genes into breeding programs is an effective strategy to combat this disease.Understanding the genetic variants that confer resistance is crucial to this strategy.The gene Bsr-d1 encodes a C2H2-like transcription factor,and its recessive allele confers broad-spectrum resistance against infections by various strains of M.oryzae.In this study,we investigated the molecular evolution of the rice blast resistance gene bsr-d1 in a representative population consisting of 827 cultivated and wild rice accessions.Our results revealed that wild rice exhibited significantly higher nucleotide diversity,with polymorphic regions primarily concentrated in the promoter region,in contrast to indica and japonica rice varieties.The Bsr-d1 gene displayed significant differentiation between indica and japonica rice varieties,with the bsr-d1 resistance allele being unique to indica rice.Haplotype network and phylogenetic analyses suggested that the bsr-d1 resistance allele most likely originated from Oryza nivara in the region adjacent to the Indian Peninsula and the Indochina Peninsula.Moreover,we explored the utilization of bsr-d1 resistance alleles in China and designed a pair of DNA primers based on the polymorphic sites for the detection of the bsr-d1 resistance gene.In summary,our study uncovering the origin and evolution of bsr-d1 will enhance our understanding of resistance gene variation and expedite the resistance breeding process.

Prox1 Suppresses Proliferation and Drug Resistance of Retinoblastoma Cells via Targeting Notch1

Objective Retinoblastoma(RB)is a prevalent type of eye cancer in youngsters.Prospero homeobox 1(Prox1)is a homeobox transcriptional repressor and downstream target of the proneural gene that is relevant in lymphatic,hepatocyte,pancreatic,heart,lens,retinal,and cancer cells.The goal of this study was to investigate the role of Prox1 in RB cell proliferation and drug resistance,as well as to explore the underlying Notch1 mechanism.Methods Human RB cell lines(SO-RB50 and Y79)and a primary human retinal microvascular endothelial cell line(ACBRI-181)were used in this study.The expression of Prox1 and Notch1 mRNA and protein in RB cells was detected using quantitative real time-polymerase chain reaction(RT-qPCR)and Western blotting.Cell proliferation was assessed after Prox1 overexpression using the Cell Counting Kit-8 and the MTS assay.Drug-resistant cell lines(SO-RB50/vincristine)were generated and treated with Prox1 to investigate the role of Prox1 in drug resistance.We employed pcDNA-Notch1 to overexpress Notch1 to confirm the role of Notch1 in the protective function of Prox1.Finally,a xenograft model was constructed to assess the effect of Prox1 on RB in vivo.Results Prox1 was significantly downregulated in RB cells.Overexpression of Prox1 effectively decreased RB cell growth while increasing the sensitivity of drug-resistant cells to vincristine.Notch1 was involved in Prox1’s regulatory effects.Notch1 was identified as a target gene of Prox1,which was found to be upregulated in RB cells and repressed by increased Prox1 expression.When pcDNA-Notch1 was transfected,the effect of Prox1 overexpression on RB was removed.Furthermore,by downregulating Notch1,Prox1 overexpression slowed tumor development and increased vincristine sensitivity in vivo.Conclusion These data show that Prox1 decreased RB cell proliferation and drug resistance by targeting Notch1,implying that Prox1 could be a potential therapeutic target for RB.

Identification of TNFRSF1A as a novel regulator of carfilzomib resistance in multiple myeloma

Multiple myeloma(MM)is a hematological tumor with high mortality and recurrence rate.Carfilzomib is a new-generation proteasome inhibitor that is used as the first-line therapy for MM.However,the development of drug resistance is a pervasive obstacle to treating MM.Therefore,elucidating the drug resistance mechanisms is conducive to the formulation of novel therapeutic therapies.To elucidate the mechanisms of carfilzomib resistance,we retrieved the GSE78069 microarray dataset containing carfilzomib-resistant LP-1 MM cells and parental MM cells.Differential gene expression analyses revealed major alterations in the major histocompatibility complex(MHC)and cell adhesion molecules.The upregulation of the tumor necrosis factor(TNF)receptor superfamily member 1A(TNFRSF1A)gene was accompanied by the downregulation of MHC genes and cell adhesion molecules.Furthermore,to investigate the roles of these genes,we established a carfilzomib-resistant cell model and observed that carfilzomib resistance induced TNFRSF1A overexpression and TNFRSF1A silencing reversed carfilzomib resistance and reactivated the expression of cell adhesion molecules.Furthermore,TNFRSF1A silencing suppressed the tumorigenesis of MM cells in immunocompetent mice,indicating that TNFRSF1A may lead to carfilzomib resistance by dampening antitumor immunity.Furthermore,our results indicated that TNFRSF1A overexpression conferred carfilzomib resistance in MM cells and suppressed the expression of MHC genes and cell adhesion molecules.The suppression of MHC genes and cell adhesion molecules may impair the interaction between immune cells and cancer cells to impair antitumor immunity.Future studies are warranted to further investigate the signaling pathway underlying the regulatory role of TNFRSF1A in MM cells.

PPP1R14A is Associated with Immunotherapy Resistance in Head and Neck Squamous Cell Carcinoma Identified by Single-Cell and Bulk RNA-Sequencing

Objective To identify nivolumab resistance-related genes in patients with head and neck squamous cell carcinoma(HNSCC)using single-cell and bulk RNA-sequencing data.Methods The single-cell and bulk RNA-sequencing data downloaded from the Gene Expression Omnibus database were analyzed to screen out differentially expressed genes(DEGs)between nivolumab resistant and nivolumab sensitive patients using R software.The Least Absolute Shrinkage Selection Operator(LASSO)regression and Recursive Feature Elimination(RFE)algorithm were performed to identify key genes associated with nivolumab resistance.Functional enrichment of DEGs was analyzed with Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analyses.The relationships of key genes with immune cell infiltration,differentation trajectory,dynamic gene expression profiles,and ligand-receptor interaction were explored.Results We found 83 DEGs.They were mainly enriched in T-cell differentiation,PD-1 and PD-L1 checkpoint,and T-cell receptor pathways.Among six key genes identified using machine learning algorithms,only PPP1R14A gene was differentially expressed between the nivolumab resistant and nivolumab sensitive groups both before and after immunotherapy(P<0.05).The high PPP1R14A gene expression group had lower immune score(P<0.01),higher expression of immunosuppressive factors(such as PDCD1,CTLA4,and PDCD1LG2)(r>0,P<0.05),lower differentiation of infiltrated immune cells(P<0.05),and a higher degree of interaction between HLA and CD4(P<0.05).Conclusions PPP1R14A gene is closely associated with resistance to nivolumab in HNSCC patients.Therefore,PPP1R14A may be a target to ameliorate nivolumab resistance of HNSCC patients.

Roles of the tumor microenvironment in the resistance to programmed cell death protein 1 inhibitors in patients with gastric cancer

Despite the continuous developments and advancements in the treatment of gastric cancer(GC),which is one of the most prevalent types of cancer in China,the overall survival is still poor for most patients with advanced GC.In recent years,with the progress in tumor immunology research,attention has shifted toward immunotherapy as a therapeutic approach for GC.Programmed cell death protein 1(PD-1)inhibitors,as novel immunosuppressive medications,have been widely utilized in the treatment of GC.However,many patients are still resistant to PD-1 inhibitors and experience recurrence in the advanced stages of PD-1 immunotherapy.To reduce the occurrence of drug resistance and recurrence in GC patients receiving PD-1 immunotherapy,to maximize the clinical activity of immunosuppressive drugs,and to elicit a lasting immune response,it is essential to research the tumor microenvironment mechanisms leading to PD-1 inhibitor resistance in GC patients.This article reviews the progress in studying the factors influencing the resistance to PD-1 inhibitors in the GC tumor microenvironment,aiming to provide insights and a basis for reducing resistance to PD-1 inhibitors for GC patients in the future.

KAT7/HMGN1 signaling epigenetically induces tyrosine phosphorylation-regulated kinase 1A expression to ameliorate insulin resistance in Alzheimer’s disease

BACKGROUND Epidemiological studies have revealed a correlation between Alzheimer’s disease(AD)and type 2 diabetes mellitus(T2D).Insulin resistance in the brain is a common feature in patients with T2D and AD.KAT7 is a histone acetyltransferase that participates in the modulation of various genes.AIM To determine the effects of KAT7 on insulin patients with AD.METHODS APPswe/PS1-dE9 double-transgenic and db/db mice were used to mimic AD and diabetes,respectively.An in vitro model of AD was established by Aβstimulation.Insulin resistance was induced by chronic stimulation with high insulin levels.The expression of microtubule-associated protein 2(MAP2)was assessed using immunofluorescence.The protein levels of MAP2,Aβ,dual-specificity tyrosine phosphorylation-regulated kinase-1A(DYRK1A),IRS-1,p-AKT,total AKT,p-GSK3β,total GSK3β,DYRK1A,and KAT7 were measured via western blotting.Accumulation of reactive oxygen species(ROS),malondialdehyde(MDA),and SOD activity was measured to determine cellular oxidative stress.Flow cytometry and CCK-8 assay were performed to evaluate neuronal cell death and proliferation,respectively.Relative RNA levels of KAT7 and DYRK1A were examined using quantitative PCR.A chromatin immunoprecipitation assay was conducted to detect H3K14ac in DYRK1A.RESULTS KAT7 expression was suppressed in the AD mice.Overexpression of KAT7 decreased Aβaccumulation and MAP2 expression in AD brains.KAT7 overexpression decreased ROS and MDA levels,elevated SOD activity in brain tissues and neurons,and simultaneously suppressed neuronal apoptosis.KAT7 upregulated levels of p-AKT and p-GSK3βto alleviate insulin resistance,along with elevated expression of DYRK1A.KAT7 depletion suppressed DYRK1A expression and impaired H3K14ac of DYRK1A.HMGN1 overexpression recovered DYRK1A levels and reversed insulin resistance caused by KAT7 depletion.CONCLUSION We determined that KAT7 overexpression recovered insulin sensitivity in AD by recruiting HMGN1 to enhance DYRK1A acetylation.Our findings suggest that KAT7 is a novel and promising therapeutic target for the resistance in AD.

Insulin-like growth factor 2 targets IGF1R signaling transduction to facilitate metastasis and imatinib resistance in gastrointestinal stromal tumors

BACKGROUND Gastrointestinal stromal tumors(GISTs)are typical gastrointestinal tract neoplasms.Imatinib is the first-line therapy for GIST patients.Drug resistance limits the long-term effectiveness of imatinib.The regulatory effect of insulin-like growth factor 2(IGF2)has been confirmed in various cancers and is related to resistance to chemotherapy and a worse prognosis.AIM To further investigate the mechanism of IGF2 specific to GISTs.METHODS IGF2 was screened and analyzed using Gene Expression Omnibus(GEO:GSE225819)data.After IGF2 knockdown or overexpression by transfection,the phenotypes(proliferation,migration,invasion,apoptosis)of GIST cells were characterized by cell counting kit 8,Transwell,and flow cytometry assays.We used western blotting to evaluate pathway-associated and epithelial-mesenchymal transition(EMT)-associated proteins.We injected transfected cells into nude mice to establish a tumor xenograft model and observed the occurrence and metastasis of GIST.RESULTS Data from the GEO indicated that IGF2 expression is high in GISTs,associated with liver metastasis,and closely related to drug resistance.GIST cells with high expression of IGF2 had increased proliferation and migration,invasiveness and EMT.Knockdown of IGF2 significantly inhibited those activities.In addition,OEIGF2 promoted GIST metastasis in vivo in nude mice.IGF2 activated IGF1R signaling in GIST cells,and IGF2/IGF1R-mediated glycolysis was required for GIST with liver metastasis.GIST cells with IGF2 knockdown were sensitive to imatinib treatment when IGF2 overexpression significantly raised imatinib resistance.Moreover,2-deoxy-D-glucose(a glycolysis inhibitor)treatment reversed IGF2 overexpressionmediated imatinib resistance in GISTs.CONCLUSION IGF2 targeting of IGF1R signaling inhibited metastasis and decreased imatinib resistance by driving glycolysis in GISTs.

NQO1 Mediates Lenvatinib Resistance by Regulating ROS-induced Apoptosis in Hepatocellular Carcinoma

Objective Hepatocellular carcinoma(HCC)is the third leading cause of cancer-associated death worldwide.As a first-line drug for advanced HCC treatment,lenvatinib faces a significant hurdle due to the development of both intrinsic and acquired resistance among patients,and the underlying mechanism remains largely unknown.The present study aims to identify the pivotal gene responsible for lenvatinib resistance in HCC,explore the potential molecular mechanism,and propose combinatorial therapeutic targets for HCC management.Methods Cell viability and colony formation assays were conducted to evaluate the sensitivity of cells to lenvatinib and dicoumarol.RNA-Seq was used to determine the differences in transcriptome between parental cells and lenvatinib-resistant(LR)cells.The upregulated genes were analyzed by GO and KEGG analyses.Then,qPCR and Western blotting were employed to determine the relative gene expression levels.Afterwards,the intracellular reactive oxygen species(ROS)and apoptosis were detected by flow cytometry.Results PLC-LR and Hep3B-LR were established.There was a total of 116 significantly upregulated genes common to both LR cell lines.The GO and KEGG analyses indicated that these genes were involved in oxidoreductase and dehydrogenase activities,and reactive oxygen species pathways.Notably,NAD(P)H:quinone oxidoreductase 1(NQO1)was highly expressed in LR cells,and was involved in the lenvatinib resistance.The high expression of NQO1 decreased the production of ROS induced by lenvatinib,and subsequently suppressed the apoptosis.The combination of lenvatinib and NQO1 inhibitor,dicoumarol,reversed the resistance of LR cells.Conclusion The high NQO1 expression in HCC cells impedes the lenvatinib-induced apoptosis by regulating the ROS levels,thereby promoting lenvatinib resistance in HCC cells.

N6-methyladenosine-modified long non-coding RNA KIF9-AS1 promotes stemness and sorafenib resistance in hepatocellular carcinoma by upregulating SHOX2 expression

BACKGROUND Hepatocellular carcinoma(HCC)is a prevalent and aggressive tumor.Sorafenib is the first-line treatment for patients with advanced HCC,but resistance to sorafenib has become a significant challenge in this therapy.Cancer stem cells play a crucial role in sorafenib resistance in HCC.Our previous study revealed that the long non-coding RNA(lncRNA)KIF9-AS1 is an oncogenic gene in HCC.However,the role of KIF9-AS1 in drug resistance and cancer stemness in HCC remains unclear.Herein,we aimed to investigate the function and mechanism of the lncRNA KIF9-AS1 in cancer stemness and drug resistance in HCC.AIM To describe the role of the lncRNA KIF9-AS1 in cancer stemness and drug resistance in HCC and elucidate the underlying mechanism.METHODS Tumor tissue and adjacent non-cancerous tissue samples were collected from HCC patients.Sphere formation was quantified via a tumor sphere assay.Cell viability,proliferation,and apoptosis were evaluated via Cell Counting Kit-8,flow cytometry,and colony formation assays,respectively.The interactions between the lncRNA KIF9-AS1 and its downstream targets were confirmed via RNA immunoprecipitation and coimmunoprecipitation.The tumorigenic role of KIF9-AS1 was validated in a mouse model.RESULTS Compared with that in normal controls,the expression of the lncRNA KIF9-AS1 was upregulated in HCC tissues.Knockdown of KIF9-AS1 inhibited stemness and attenuated sorafenib resistance in HCC cells.Mechanistically,N6-methyladenosine modification mediated by methyltransferase-like 3/insulin-like growth factor 2 mRNA-binding protein 1 stabilized and increased the expression of KIF9-AS1.Additionally,KIF9-AS1 increased the stability and expression of short stature homeobox 2 by promoting ubiquitin-specific peptidase 1-induced deubiquitination.Furthermore,depletion of KIF9-AS1 alleviated sorafenib resistance in a xenograft mouse model of HCC.CONCLUSION The N6-methyladenosine-modified lncRNA KIF9-AS1 promoted stemness and sorafenib resistance in HCC by upregulating short stature homeobox 2 expression.

Studies on the temporal,structural,and interacting features of the clubroot resistance gene Rcr1 using CRISPR/Cas9-based systems

Clubroot disease is a severe threat to Brassica crops globally,particularly in western Canada.Genetic resistance,achieved through pyramiding clubroot resistance(CR)genes with different modes of action,is the most important strategy for managing the disease.However,studies on the CR gene functions are quite limited.In this study,we have conducted investigations into the temporal,structural,and interacting features of a newly cloned CR gene,Rcr1,using CRISPR/Cas9 technology.For temporal functionality,we developed a novel CRISPR/Cas9-based binary vector,pHHIGR-Hsp18.2,to deliver Rcr1 into a susceptible canola line(DH12075)and observed that early expression of Rcr1 is critical for conferring resistance.For structural functionality,several independent mutations in specific domains of Rcr1 resulted in loss-offunction,highlighting their importance for CR phenotype.In the study of the interacting features of Rcr1,a cysteine protease gene and its homologous allele in canola were successfully disrupted via CRISPR/Cas9 as an interacting component with Rcr1 protein,resulting in the conversion from clubroot resistant to susceptible in plants carrying intact Rcr1.These results indicated an indispensable role of these two cysteine proteases in Rcr1-mediated resistance response.This study,the first of its kind,provides valuable insights into the functionality of Rcr1.Further,the new vector p HHIGR-Hsp18.2 demonstrated an inducible feature on the removal of add-on traits,which should be useful for functional genomics and other similar research in brassica crops.

Ghrelin regulates insulin resistance by targeting insulin-like growth factor-1 receptor via miR-455-5p in hepatic cells

Objective: To explore the mechanism by which ghrelin regulates insulin sensitivity through modulation of miR-455-5p in hepatic cells. Methods: HepG2 cells were treated with or without DAG (1 μM). Glucose consumption, intracellular glycogen content, phosphorylation of PI3K and Akt stimulated by insulin, expression of miR-455-5p, as well as IGF-1R protein level were analyzed. In addition, bioinformatic analysis, dual luciferase reporter assay, miR- 455-5p mimic or inhibitor treatment was conducted to investigate the molecular mechanisms. Results: High glucose treatment upregulated miR-455-5p expression but reduced glucose consumption and glycogen content. DAG reversed the effect of high glucose on glucose metabolism, increased protein level of IGF-1R and phosphorylation of PI3K/Akt stimulated by insulin, as well as downregulated miR-455-5p expression. Bioinformatic analysis indicated IGF-1R was the target of miR-455-5p. Dual luciferase reporter assay, as well as transfection with miR-455-5p mimic/inhibitor confirmed that DAG activated IGF-1R/PI3K/Akt signaling via inhibiting miR-455-5p. Conclusion: DAG improves insulin resistance via miR-455-5p- mediated activation of IGF-1R/PI3K/Akt system, suggesting that suppression of miR-455-5p or activation of DAG may be potential targets for T2DM therapy.

Reversal of Multi-Drug Resistance by Vector-Based-ShRNA-Mdr1 In Vitro and In Vivo

In order to investigate the effects of vector-based hairpin small interference RNA （shRNA） on the reversal of multi-drug resistance （mdr） of A2780/Taxol cells, a novel vector pEGFP-HI/mdrl containing mdrl-shRNA targeting at position 2943-2963 of mdrl was designed and synthesized. Subsequently, A2780/Taxol cells were transfected with pEGFP-H1/rndrl, and the expression ofmdrl mRNA and P-gp was detected by using RT-PCR and Western blot respectively. MTT was used to measure the 50% inhibition concentration （IC50） of Taxol to A2780/Taxol cells. The results showed that at the 24th and 48th h after transfection, the expression of mdrl mRNA was decreased to （52.1±1.0）% and （0.01±1.7）%, and that of P-gp decreased to （88.3±2.1）% and 0%, respectively. At the 48th h after transfection, the relative reversal rate of A2780/Taxol cells to Taxol was 69.54%. In vivo, the nude mice xenografts were injected with pEGFP-H1/mdrl, and then administrated Taxol. The tumor volume in pEGFP-H1/mdrl-transfected group was significantly reduced as compared with that in blank control group or pEGFP-Hl-transfected group （807.20±103.16 vs 1563.78±210.54 or 1480.78±241.24 mm^3, both P〈0.01）. These results suggested that transfection of pEGFP-HI/mdrl could efficiently down-regulate the expression of mdrl mRNA and P-gp in A2780/Taxol cells, and effectively restore the sensitivity of A2780/Taxol ceils to Taxol both in vitro and in vivo.

Microstructure,mechanical properties and creep resistance of Mg-(8%-12%)Zn-(2%-6%) Al alloys

The microstructural characteristics, mechanical properties and creep resistance of Mg-（8%-12%） Zn-（2%-6%） A1 alloys were investigated to get a better overall understanding of these series alloys. The results indicate that the microstructure of the alloys ZA82, ZA102 and ZA122 with the mass ratio of Zn to A1 of 4-6 is mainly composed of a-Mg matrix and two different morphologies of precipitates （block τ-Mg32（Al, Zn）49 and dense lamellar ε-Mg51Zn20）, the alloys ZA84, ZA104 and ZA124 with the mass ratio of 2-3 contain α-Mg matrix and only block r phases, and the alloys ZA86, ZA106 and ZA126 with the mass ratio of 1-2 consist of a-Mg matrix, block r precipitates, lamellar Ф-Al2Mg5Zn2 eutectics and flocculent β-Mg17Al12 compounds. The alloys studied with the mass ratio of Zn to A1 of 2-3 exhibit high creep resistance, and the alloy ZA124 with the continuous network of r precipitating along grain boundaries shows the highest creep resistance.

Role of Hypoxia-inducible Factor-1α in Formation of Muttidrug Resistance Induced by Microenvironment in Hepatocellular Carcinoma

Objective： To explore the role of hypoxia-inducible factor-1α （HIF-1α） in formation of multidrug resistance （MDR） induced by microenvironment and to find a new and effective molecular target on preventing and reversing chemoresistance in hepatocellular carcinoma （HCC）. Methods： In HepG2 cells exposed to hypoxia, low glucose or transfected by plasmid pcDNA3/HBX, the expression of HIF-1α mRNA and protein was respectively detected using real-time fluorescent quantitative PCR and Westernblot technique and its expression localization was investigated by immunocytochemical technique. Plasmid pcDNA3/HIF-1α was transfected into HepG2 cells and then the expression of multidrug resistance related genes mdrl, multidrug resistance-associated protein 1 （MRP1） and lung resistance protein （LRP） in transfected cells was determined by the same methods. Results： In HepG2 cells respectively exposed to hypoxia, low glucose or transfected by plasmid pcDNA3/HBX, HIF-1α was overexpressed at mRNA and protein levels to varying degrees and translocated into nucleus. The gene expression levels of mdrl, MRP1 and LRP in HepG2 cells transfected by plasmid pcDNA3/HIF-1α were respectively increased by 2.4±0.2, 2.2±0.3 and 2.3±0.4 folds as compared with those in non-transfected HepG2 cells （all P〈0.01） and similar changes were observed in protein level. Conclusion： Microenvironmental factors around HCC could modulate the transcription of the MDR related genes by nuclear transcript factor HIF-1α, thereby conferred MDR of HCC. Up-regulation of HIF-1α expression could hold a central position in the formation of MDR of HCC induced by microenvironment. HIF-1α probably becomes a new and effective molecular target on preventing and reversing MDR in HCC.

Long interspersed nuclear element ORF-1 protein promotes proliferation and resistance to chemotherapy in hepatocellular carcinoma

AIM:To clarify the specific roles and mechanisms of long interspersed nuclear element-1 ORF-1 protein [human long interspersed nuclear element-1(LINE-1),ORF-1p] in chemotherapeutic drug resistance and cell proliferation regulation in hepatocellular carcinoma(HCC) cells.METHODS:MTT assays were performed to identify the effect of the chemotherapeutic drug toxicity on HepG2 cells.Cell proliferation inhibition and the IC 50 were calculated by the Origin 8.0 software.Western blotting assays were performed to investigate whether LINE-1 ORF-1p modulates the expression of some important genes,including p53,p27,p15,Bcl-2,mdr,and p-gp.To corroborate the proliferation and anchor-independent growth results,the HepG2 cells were analyzed by flow cytometry to investigate the effect of LINE-1 ORF1p on the apoptosis regulation.RESULTS:LINE-1 ORF-1p contributed to the resistance to several chemotherapeutic drugs(cisplatin and epirubicin) in HepG2 cells.The IC 50 of the epirubicin and cisplatin increased from 36.04 nmol/L to 59.11 nmol/L or from 37.94 nmol/L to 119.32 nmol/L.Repression of LINE-1 ORF-1p expression by the siRNA could markedly enhance the response of HepG2 cells to the epirubicin and cisplatin.The IC 50 correspondingly decreased from 28.06 nmol/L to 3.83 nmol/L or from 32.04 nmol/L to 2.89 nmol/L.Interestingly,down-regulation of LINE-1 ORF-1p level by siRNA could promote the response of HepG2 cells to the paclitaxel.The IC 50 decreased from 35.90 nmol/L to 7.36 nmol/L.However,overexpression of LINE-1 ORF-1p did not modulate the paclitaxel toxicity in HepG2 cells.Further Western blotting revealed that LINE-1 ORF-1p enhanced mdr and p-gp gene expression.As a protein arrested in the nucleus,LINE-1 ORF-1p may function through modulating transcriptional activity of some important transcription factors.Indeed,LINE-1 ORF-1p promoted HepG2 cell proliferation,anchor-independent growth and protected the cells against apoptosis through modulating the expression of p15,p21,p53,and Bcl-2 genes.CONCLUSION:LINE-1 ORF-1p promotes HepG2 cell proliferation and plays an important role in the resistance of chemotherapeutic drugs.By establishing novel roles and defining the mechanisms of LINE-1 ORF1p in HCC chemotherapeutic drug resistance and cell proliferation regulation,this study indicates that LINE-1 ORF-1p is a potential target for overcoming HCC chemotherapeutic resistance.

The Roles of Four Multi-drug Resistance Proteins in Hepatocellular Carcinoma Multidrug Resistance

The roles of multi-drug resistance protein 1 （MDR1）, multi-drug resistance related protein 1 （MRP1）, lung resistance protein （LRP） and breast cancer resistance protein （BCRP） in the multi-drug resistance （MDR） of hepatocellular carcinoma （HCC） were studied. By exposing HepG2 cell line to progressively increased concentrations of adriamycin （ADM）, HepG2 multi-drug resistant subline （HepG2/ADM） was induced. The MDR index of HepG2/ADM was detected by using MTT. The expressions of the four MDR proteins in the three cell lines （L02, HepG2, HepG2/ADM） were investigated at mRNA and protein levels by real-time RT-PCR and Western blot respectively. Our results showed that when the ADM concentration was under 100 pg/L, HepG2 could easily be induced to be drug-resistant. The IC50 of the HepG2/ADM to ADM was 282 times that of HepG2. The expression of MDR1 and BCRP mRNA in HepG2/ADM cells were 400 and 9 times that of HepG2 cells respectively while there was no difference in the mRNA expressions of MRPl and LRE There was no difference between HepG2 and L02 cells in the mRNA expressions of the four genes. At the protein level, the expressions of MDRI, BCRP and LRP but MRPl in HepG2/ADM were significantly higher than those of HepG2 and L02. Between HepG2 and L02, there was no difference in the expressions of four genes at the protein level. HepG2/ADM is a good model for the study of MDR. The four genes are probably the normally expressed gene in liver. The expressions of MDRl and BCRP could be up-regulated by anti-cancer agents in vitro. The MDR of HCC was mainly due to the up-regulation of MDR1 and BCRP but MRP1 and LRE These findings suggest they may serve as targets for the reversal of MDR of HCC.

Synergistic Effect of Hyperthermia and Neferine on Reverse Multidrug Resistance in Adriamycin-resistant SGC7901/ADM Gastric Cancer Cells

Multidrug resistance（MDR） plays a major obstacle to successful gastric cancer chemotherapy.The purpose of this study was to investigate the MDR reversal effect and mechanisms of hyperthermia in combination with neferine（Nef） in adriamycin（ADM） resistant human SGC7901/ADM gastric cancer cells.The MDR cells were heated at 42℃ and 45℃ for 30 min alone or combined with 10 μg/mL Nef.The cytotoxic effect of ADM was evaluated by MTT assay.Cellular plasma membrane lipid fluidity was detected by fluorescence polarization technique.Intracellular accumulation of ADM was monitored with high performance liquid chromatography.Mdr-1 mRNA,P-glycoprotein（P-gp）,γH2AX expression and γH2AX foci formation were determined by real-time PCR,Western blot and immunocytochemical staining respectively.It was found that different heating methods induced different cytotoxic effects.Water submerged hyperthermia had the strongest cytotoxicity of ADM and Nef combined with hyperthermia had a synergistic cytotoxicity of ADM in the MDR cells.The water submerged hyperthermia increased the cell membrane fluidity.Both water submerged hyperthermia and Nef increased the intracellular accumulation of ADM.The water submerged hyperthermia and Nef down-regulated the expression of mdr-1 mRNA and P-gp.The water submerged hyperthermia could damage DNA and increase the γH2AX expression of SGC7901/ADM cells.The higher temperature was,the worse effect was.Our results show that combined treatment of hyperthermia with Nef can synergistically reverse MDR in human SGC7901/ADM gastric cancer cells.

Relationship between Methylation Status of Multi-drug Resistance Protein(MRP) and Multi-drug Resistance in Lung Cancer Cell Lines

Objective： To study the relationship between the methylation status of multi-drug resistance protein （MRP） gene and the expression of its mRNA and protein in lung cancer cell lines. Methods： Human embryo lung cell line WI-38, lung adenocarcinoma cell line SPCA-1 and its drug-resistant cells induced by different concentrations of doxorubicin were treated with restriction endonuclease Eco47III. The methylation status of MRP was examined by PCR, and the expressions of its mRNA and protein were evaluated by in situ hybridization and immunohistochemistry. Results： MRP gene promoter region of WI-38 cells was in hypermethylation status, but the promoter region of MRP in SPCA-1 cells and their resistant derivatives induced by different concentrations of doxorubicin were in hypomethylation status. There were significant differences in the expression of MRP mRNA among WI-38 cell line, SPCA-1 cells and their drug-resistant derivatives induced by different concentration of doxorubicin. Consistently, MRP immunostaining presented similar significant differences. Conclusion： The promoter region of MRP in SPCA-1 lung adenocarcinoma cells was in hypomethylation status. The hypomethylation status of 5＇ regulatory region of MRP promoter is an important structural basis that can increase the activity of transcription and results in the development of drug resistance in lung cancer.