BACKGROUND: Drug resistance is the main cause of high mortality of lung cancer. This study was conducted to investigate the effect of folic acid (FA) on the resistance of non-small cell lung cancer (NSCLC) cells to Osimertinib (OSM) by regulating the methylation of dual specificity phosphatase 1 (DUSP1). METHODS: The OSM resistant NSCLC cell line PC9R was establishd by gradually escalation of OSM concentration in PC9 cells. PC9R cells were randomly grouped into Control group, OSM group (5 μmol/L OSM), FA group (600 nmol/L FA), methylation inhibitor decitabine (DAC) group (10 μmol/L DAC), FA+OSM group (600 nmol/L FA+5 μmol/L OSM), and FA+OSM+DAC group (600 nmol/L FA+5 μmol/L OSM+10 μmol/L DAC). CCK-8 method was applied to detect cell proliferation ability. Scratch test was applied to test the ability of cell migration. Transwell assay was applied to detect cell invasion ability. Flow cytometry was applied to measure and analyze the apoptosis rate of cells in each group. Real-time fluorescence quantitative polymerase chain reaction (RT-qPCR) method was applied to detect the expression level of DUSP1 mRNA in cells. Methylation specific PCR (MSP) was applied to detect the methylation status of the DUSP1 promoter region in each group. Western blot was applied to analyze the expression levels of DUSP1 protein and key proteins in the DUSP1 downstream mitogen-activated protein kinase (MAPK) signaling pathway in each group. RESULTS: Compared with the Control group, the cell OD450 values (48 h, 72 h), scratch healing rate, number of cell invasions, and expression of DUSP1 in the OSM group were obviously decreased (P<0.05); the apoptosis rate, the methylation level of DUSP1, the expression of p38 MAPK protein, and the phosphorylation level of extracellular regulated protein kinases (ERK) were obviously increased (P<0.05); the cell OD450 values (48, 72 h), scratch healing rate, number of cell invasions, and expression of DUSP1 in the DAC group were obviously increased (P<0.05); the apoptosis rate, the expression of p38 MAPK protein, the phosphorylation level of ERK, and the methylation level of DUSP1 were obviously reduced (P<0.05). Compared with the OSM group, the cell OD450 values (48, 72 h), scratch healing rate, number of cell invasions, and expression of DUSP1 in the FA+OSM group were obviously decreased (P<0.05); the apoptosis rate, the methylation level of DUSP1, the expression of p38 MAPK protein, and the phosphorylation level of ERK were obviously increased (P<0.05). Compared with the FA+OSM group, the cell OD450 values (48, 72 h), scratch healing rate, number of cell invasions, and expression of DUSP1 in the FA+OSM+DAC group were obviously increased; the apoptosis rate, the methylation level of DUSP1, the expression of p38 MAPK protein, and the phosphorylation level of ERK were obviously reduced (P<0.05). CONCLUSIONS FA may inhibit DUSP1 expression by enhancing DUSP1 methylation, regulate downstream MAPK signal pathway, then promote apoptosis, inhibit cell invasion and metastasis, and ultimately reduce OSM resistance in NSCLC cells.
Humans ; Carcinoma, Non-Small-Cell Lung/genetics* ; Lung Neoplasms/genetics* ; Dual Specificity Phosphatase 1/pharmacology* ; Cell Proliferation ; p38 Mitogen-Activated Protein Kinases/pharmacology* ; Methylation ; Apoptosis ; Cell Line, Tumor

This study aims to decipher the mechanism underlying the effect of Shaofu Zhuyu Decoction on endometriosis(EMT)-associated dysmenorrhea in rats with the syndrome of cold coagulation and blood stasis based on mitogen-and stress-activated protein kinase 1/2(MSK1/2).We employed a random number table to randomly assign SPF female non-pregnant rats into the sham group, and treated the rest rats with autologous transplantation+refrigerator freezing for the modeling of the syndrome of cold coagulation and blood stasis.The modeled rats were then randomly assigned into the control group and high-, medium-and low-dose Shaofu Zhuyu Decoction groups.The rats in the low-, medium-, and high-dose decoction groups were respectively administrated with 9, 4.5, and 2.3 g·kg~(-1) decoction through gavage once a day for 2 consecutive weeks, and those in the control group were administrated with 0.24 mg·kg~(-1) gestrinone through gavage once every 3 days for 2 weeks.After that, the size of ectopic focus in each rat was measured via laparotomy.Enzyme-linked immunosorbent assay(ELISA) was adopted to determine the expression of interleukin(IL)-6, IL-10, prostaglandin E2(PGE2), tumor necrosis factor-α(TNF-α).Western blot was employed to determine the protein levels of MSK1/2 and dual-specificity phosphatase 1(DUSP1) and real-time quantitative polymerase chain reaction(RT-PCR) to determine the mRNA levels of the two genes in rat eutopic endometrial tissue.Compared with the sham group, the model group showed increased levels of IL-6, PGE2, and TNF-α while decrease level of IL-10 in the serum(P<0.01).Compared with the model group, the high-and medium-dose decoction groups and the gestrinone group had declined levels of IL-6, PGE2, and TNF-α while risen level of IL-10 in the serum(P<0.01).The model group had lower protein levels and mRNA levels of MSK1/2 and DUSP1 in the eutopic endometrial tissue than the sham group(P<0.01). The high-and medium-dose decoction groups and the gestrinone group had higher protein and mRNA levels of MSK1/2 and DUSP1 in the eutopic endometrial tissue than the model group(P<0.01).The results indicated that Shaofu Zhuyu Decoction can regulate the abnormal expression of pro-inflammatory cytokines TNF-α, IL-6, and PGE2 and anti-inflammatory cytokines IL-10 and DUSP1 via MSK1/2 to alleviate EMT-associated dysmenorrhea in rats with the syndrome of cold coagulation and blood stasis.
Animals ; Female ; Rats ; Anti-Inflammatory Agents/therapeutic use* ; Cytokines ; Dinoprostone ; Drugs, Chinese Herbal/therapeutic use* ; Dual-Specificity Phosphatases ; Dysmenorrhea/genetics* ; Endometriosis/genetics* ; Gestrinone/therapeutic use* ; Interleukin-10 ; Interleukin-6 ; Mitogen-Activated Protein Kinase 8/therapeutic use* ; Mitogens/therapeutic use* ; RNA, Messenger ; Tumor Necrosis Factor-alpha/metabolism*

OBJECTIVE: To investigate the expression of CDC25A in non- small cell lung cancer (NSCLC) tissues and explore its correlation with the clinicpathological features of the patients and the expressions of let-7a1 and let-7c. METHODS: We collected surgical specimens of pathologically confirmed NSCLC tissues and paired adjacent lung tissues from 44 patients and tissues of benign lung lesions from 9 patients. The expressions of CDC25A protein and mRNA in the tissues were detected by immunohistochemistry and fluorescence quantitative RT-PCR, respectively; the expressions of let-7a1 and let-7c mRNA were detected using tail-adding fluorescence quantitative RT-PCR. RESULTS: The positivity rate of CDC25A protein expression was significantly higher in NSCLC tissues than in the adjacent tissues and benign pulmonary lesions ( CONCLUSIONS The expression level of CDC25A is significantly increased in NSCLC with a negative correlation with Let-7c expression, which identifies CDC25A as a possible downstream target gene of Let-7c.
Carcinoma, Non-Small-Cell Lung/genetics* ; Gene Expression Regulation, Neoplastic ; Humans ; Lung ; Lung Neoplasms/genetics* ; Lymphatic Metastasis ; MicroRNAs ; RNA, Messenger/genetics* ; cdc25 Phosphatases

OBJECTIVE: To assess the association of single nucleotide polymorphisms (SNPs) of dual specificity phosphatase 9 (DUSP9) gene rs5945326 locus with gestational diabetes mellitus (GDM). METHODS: Genotypes for the rs5945326 locus were determined for 206 pregnant women with GDM (GDM group) and 189 unaffected pregnant women (control group). Allelic and genotypic frequencies of the GDM and control groups were compared. For individuals with various genotypes, the level of blood glucose, serum lipids, and body mass index (BMI) were also compared. RESULTS: The frequencies of AA, AG and GG genotypes for the GDM group were 32.2%, 52.2% and 15.6%, respectively, and 41.2%, 43.9% and 15.0%, for the control group, respectively. No significant difference was detected in the distribution of above genotypes between the two groups (chi-square=3.601, P=0.165). The frequencies of alleles A and G were 58.3% and 41.7% in the GDM group, and 63.1% and 36.9% in the control group, respectively. No significant difference was detected between the two groups too (chi-square=1.894, P=0.188). The high density lipoprotein (HDL) levels of the GG genotype [(2.34×0.61) mmol/L] was significantly higher than that of the AG+AA genotype [(2.06×0.56) mmol/L] (t=2.993, P=0.003). No significant difference was detected in other clinical indexes between the two groups (P> 0.05). CONCLUSION The SNP rs5945326 in DUSP9 gene may be not associated with the risk of GDM. However, there are correlated with HDL levels.
Alleles ; Diabetes, Gestational ; genetics ; Dual-Specificity Phosphatases ; genetics ; Female ; Gene Frequency ; Genotype ; Humans ; Mitogen-Activated Protein Kinase Phosphatases ; genetics ; Polymorphism, Single Nucleotide ; Pregnancy

OBJECTIVETo investigate the effects of evodiamine (Evo), a component of Evodiaminedia rutaecarpa (Juss.) Benth, on cardiomyocyte hypertrophy induced by angiotensin II (Ang II) and further explore the potential mechanisms.

METHODSCardiomyocytes from neonatal Sprague Dawley rats were isolated and characterized, and then the cadiomyocyte cultures were randomly divided into control, model (Ang II 0.1 μmol/L), and Evo (0.03, 0.3, 3 μmol/L) groups. The cardiomyocyte surface area, protein level, intracellular free calcium ([Ca]) concentration, activity of nitric oxide synthase (NOS) and content of nitric oxide (NO) were measured, respectively. The mRNA expressions of atrial natriuretic factor (ANF), calcineurin (CaN), extracellular signal-regulated kinase-2 (ERK-2), and endothelial nitric oxide synthase (eNOS) of cardiomyocytes were analyzed by real-time reverse transcriptionpolymerase chain reaction. The protein expressions of calcineurin catalytic subunit (CnA) and mitogen-activated protein kinase phosphatase-1 (MKP-1) were detected by Western blot analysis.

RESULTSCompared with the control group, Ang II induced cardiomyocytes hypertrophy, as evidenced by increased cardiomyocyte surface area, protein content, and ANF mRNA expression; increased intracellular free calcium ([Ca]) concentration and expressions of CaN mRNA, CnA protein, and ERK-2 mRNA, but decreased MKP-1 protein expression (P<0.05 or P<0.01). Compared with Ang II, Evo (0.3, 3 μmol/L) significantly attenuated Ang II-induced cardiomyocyte hypertrophy, decreased the [Ca] concentration and expressions of CaN mRNA, CnA protein, and ERK-2 mRNA, but increased MKP-1 protein expression (P<0.05 or P<0.01). Most interestingly, Evo increased the NOS activity and NO production, and upregulated the eNOS mRNA expression (P<0.05).

CONCLUSIONEvo signifificantly attenuated Ang II-induced cardiomyocyte hypertrophy, and this effect was partly due to promotion of NO production, reduction of [Ca]i concentration, and inhibition of CaN and ERK-2 signal transduction pathways.

Angiotensin II ; Animals ; Atrial Natriuretic Factor ; metabolism ; Calcineurin ; genetics ; metabolism ; Calcium ; metabolism ; Dual Specificity Phosphatase 1 ; genetics ; metabolism ; Extracellular Signal-Regulated MAP Kinases ; genetics ; metabolism ; Hypertrophy ; Myocytes, Cardiac ; drug effects ; metabolism ; pathology ; Nitric Oxide ; metabolism ; Nitric Oxide Synthase Type III ; metabolism ; Quinazolines ; pharmacology ; RNA, Messenger ; genetics ; metabolism ; Rats, Sprague-Dawley

PURPOSE: The cyclin-dependent kinase 1 (Cdk1) and cyclin B complex performs important roles in the transition from the G2 to M phase in the cell cycle through removal of inhibitory phosphates on Cdk1, and Cdc25B, which is a dual-specific phosphatase, mediates these dephosphorylation events. However, measuring Cdc25B activity by existing methods is hampered by inadequate nonspecific substrates and the need to use a radiolabeled isotope. The present study aimed to develop an improved method with which to properly measure Cdc25B activity using a novel nonradioisotopic assay and Cdc25B overexpression cell lines. MATERIALS AND METHODS: A nonradioisotopic Cdk1 kinase assay, based on Western blotting for retinoblastoma protein and histone H1, was used to analyze Cdc25B activity. Also, stable Cdc25B2 and Cdc25B3 overexpression HeLa cell lines were constructed using the tetracycline-regulated expression system and were applied as a tool for screening for inhibitors of Cdc25B. RESULTS: The present study developed and optimized a nonradioisotopic assay method to properly measure Cdc25B activity. Furthermore, we constructed stable Cdc25B2 and Cdc25B3 overexpression HeLa cell lines for the establishment of a strong assay system with which to evaluate the specificity of Cdc25B inhibitors under conditions similar to the intracellular environment. These methods were confirmed as useful tools for measuring Cdc25B activity. CONCLUSION: The nonradioisotopic Cdk1 kinase assay and Cdc25B overexpression cell lines developed in this study can be conveniently used as tools for screening inhibitors of Cdc25B phosphatase as anticancer drugs.
Blotting, Western ; CDC2 Protein Kinase ; cdc25 Phosphatases ; Cell Cycle ; Cell Division ; Cell Line* ; Cyclin B ; HeLa Cells ; Histones ; Humans ; Mass Screening* ; Methods ; Phosphates ; Retinoblastoma Protein ; Sensitivity and Specificity

Background: Glucocorticoid (GC) is the first-line therapy for asthma, but some asthmatics are insensitive to it. Glucocorticoid-induced transcript 1 gene (GLCCI1) is reported to be associated with GCs efficiency in asthmatics, while its exact mechanism remains unknown. Methods: A total of 30 asthmatic patients received fluticasone propionate for 12 weeks. Forced expiratory volume in 1 s (FEV) and GLCCI1 expression were detected. Asthma model was constructed in wild-type and GLCCI1 knockout (GLCCI1) mice. Glucocorticoid receptor (GR) and mitogen-activated protein kinase phosphatase 1 (MKP-1) expression were detected by polymerase chain reaction and Western blotting (WB). The phosphorylation of p38 mitogen-activated protein kinase (MAPK) was also detected by WB. Results: In asthmatic patients, the change of FEV was well positively correlated with change of GLCCI1 expression (r = 0.430, P = 0.022). In animal experiment, GR and MKP-1 mRNA levels were significantly decreased in asthmatic mice than in control mice (wild-type: GR: 0.769 vs. 1.000, P = 0.022; MKP-1: 0.493 vs. 1.000, P < 0.001. GLCCI1: GR: 0.629 vs. 1.645, P < 0.001; MKP-1: 0.377 vs. 2.146, P < 0.001). Hydroprednisone treatment significantly increased GR and MKP-1 mRNA expression levels than in asthmatic groups; however, GLCCI1 asthmatic mice had less improvement (wild-type: GR: 1.517 vs. 0.769, P = 0.023; MKP-1: 1.036 vs. 0.493, P = 0.003. GLCCI1: GR: 0.846 vs. 0.629, P = 0.116; MKP-1: 0.475 vs. 0.377, P = 0.388). GLCCI1 asthmatic mice had more obvious phosphorylation of p38 MAPK than wild-type asthmatic mice (9.060 vs. 3.484, P < 0.001). It was still higher even though after hydroprednisone treatment (6.440 vs. 2.630, P < 0.001). Conclusions: GLCCI1 deficiency in asthmatic mice inhibits the activation of GR and MKP-1 and leads to more obvious phosphorylation of p38 MAPK, leading to a decremental sensitivity to GCs. Trial Registration ChiCTR.org.cn, ChiCTR-RCC-13003634; http://www.chictr.org.cn/showproj.aspx?proj=5926.
Animals ; Asthma ; drug therapy ; metabolism ; Dual Specificity Phosphatase 1 ; genetics ; metabolism ; Forced Expiratory Volume ; genetics ; physiology ; Glucocorticoids ; therapeutic use ; Mice ; Mice, Knockout ; Phosphorylation ; genetics ; physiology ; Receptors, Glucocorticoid ; deficiency ; genetics ; metabolism ; p38 Mitogen-Activated Protein Kinases ; genetics ; metabolism

Acquisition of resistance to anti-cancer drugs is a significant obstacle to effective cancer treatment. Although several efforts have been made to overcome drug resistance in cancer cells, the detailed mechanisms have not been fully elucidated. Here, we investigated whether microRNAs (miRNAs) function as pivotal regulators in the acquisition of anti-cancer drug resistance to 5-fluorouracil (5-FU). A survey using a lentivirus library containing 572 precursor miRNAs revealed that five miRNAs promoted cell survival after 5-FU treatment in human hepatocellular carcinoma Hep3B cells. Among the five different clones, the clone expressing miR-200a-3p (Hep3B-miR-200a-3p) was further characterized as a 5-FU-resistant cell line. The cell viability and growth rate of Hep3B-miR-200a-3p cells were higher than those of control cells after 5-FU treatment. Ectopic expression of a miR-200a-3p mimic increased, while inhibition of miR-200a-3p downregulated, cell viability in response to 5-FU, doxorubicin, and CDDP (cisplatin). We also showed that dual-specificity phosphatase 6 (DUSP6) is a novel target of miR-200a-3p and regulates resistance to 5-FU. Ectopic expression of DUSP6 mitigated the pro-survival effects of miR-200a-3p. Taken together, these results lead us to propose that miR-200a-3p enhances anti-cancer drug resistance by decreasing DUSP6 expression.
Carcinoma, Hepatocellular ; Cell Line ; Cell Survival ; Clone Cells ; Doxorubicin ; Drug Resistance ; Dual Specificity Phosphatase 6* ; Dual-Specificity Phosphatases* ; Ectopic Gene Expression ; Fluorouracil* ; Humans ; Lentivirus ; MicroRNAs

BACKGROUND: Lung cancer is the leading cause of cancer-related death worldwide, for which smoking is considered as the primary risk factor. The present study was conducted to determine whether genetic alterations induced by radon exposure are associated with the susceptible risk of lung cancer in never smokers. METHODS: To accurately identify mutations within individual tumors, next generation sequencing was conduct for 19 pairs of lung cancer tissue. The associations of germline and somatic variations with radon exposure were visualized using OncoPrint and heatmap graphs. Bioinformatic analysis was performed using various tools. RESULTS: Alterations in several genes were implicated in lung cancer resulting from exposure to radon indoors, namely those in epidermal growth factor receptor (EGFR), tumor protein p53 (TP53), NK2 homeobox 1 (NKX2.1), phosphatase and tensin homolog (PTEN), chromodomain helicase DNA binding protein 7 (CHD7), discoidin domain receptor tyrosine kinase 2 (DDR2), lysine methyltransferase 2C (MLL3), chromodomain helicase DNA binding protein 5 (CHD5), FAT atypical cadherin 1 (FAT1), and dual specificity phosphatase 27 (putative) (DUSP27). CONCLUSIONS: While these genes might regulate the carcinogenic pathways of radioactivity, further analysis is needed to determine whether the genes are indeed completely responsible for causing lung cancer in never smokers exposed to residential radon.
Cadherins ; Computational Biology ; DNA-Binding Proteins ; Dual-Specificity Phosphatases ; Genes, Homeobox ; Lung Neoplasms* ; Lung* ; Lysine ; Radioactivity ; Radon* ; Receptor, Epidermal Growth Factor ; Risk Factors ; Smoke ; Smoking ; TYK2 Kinase

The mitogen-activated protein kinases (MAPKs) are key regulators of cell growth and survival in physiological and pathological processes. Aberrant MAPK signaling plays a critical role in the development and progression of human cancer, as well as in determining responses to cancer treatment. The MAPK phosphatases (MKPs), also known as dual-specificity phosphatases (DUSPs), are a family of proteins that function as major negative regulators of MAPK activities in mammalian cells. Studies using mice deficient in specific MKPs including MKP1/DUSP1, PAC-1/DUSP2, MKP2/DUSP4, MKP5/DUSP10 and MKP7/DUSP16 demonstrated that these molecules are important not only for both innate and adaptive immune responses, but also for metabolic homeostasis. In addition, the consequences of the gain or loss of function of the MKPs in normal and malignant tissues have highlighted the importance of these phosphatases in the pathogenesis of cancers. The involvement of the MKPs in resistance to cancer therapy has also gained prominence, making the MKPs a potential target for anti-cancer therapy. This review will summarize the current knowledge of the MKPs in cancer development, progression and treatment outcomes.
Animals ; Dual-Specificity Phosphatases ; Homeostasis ; Humans ; Mice ; Mitogen-Activated Protein Kinase Phosphatases* ; Mitogen-Activated Protein Kinases ; Pathologic Processes ; Phosphoric Monoester Hydrolases