Head and neck squamous cell carcinoma-specific prognostic signature and drug sensitive subtypes based on programmed cell death-related genes.

Background: As a complex group of malignancies, head and neck squamous cell carcinoma (HNSC) is one of the leading causes of cancer mortality. This study aims to establish a reliable clinical classification and gene signature for HNSC prognostic prediction and precision treatments. Methods: A consensus clustering analysis was performed to group HNSC patients in The Cancer Genome Atlas (TCGA) database based on genes linked to programmed cell death (PCD). Differentially expressed genes (DEGs) between subtypes were identified using the "limma" R package. The TCGA prognostic signature and PCD-related prognostic genes were found using a least absolute shrinkage and selection operator (LASSO) regression analysis and univariate Cox regression analysis. The robustness of the LASSO analysis was validated using datasets GSE65858 and GSE41613. A cell counting kit-8 (CCK-8) test, Western blot, and real-time reverse transcriptase-polymerase chain reaction (RT-qPCR) were used to evaluate the expression and viability of prognostic genes. Results: Four molecular subtypes were identified in PCD-related genes. Subtype C4 had the best prognosis and the highest immune score, while subtype C1 exhibited the most unfavorable outcomes. Three hundred shared DEGs were identified among the four subtypes, and four prognostic genes (CTLA4, CAMK2N1, PLAU and CALML5) were used to construct a TCGA-HNSC prognostic model. High-risk patients manifested poorer prognosis, more inflammatory pathway enrichment, and lower immune cell infiltration. High-risk patients were more prone to immune escape and were more likely to be resistant to Cisplatin and 5-Fluorouracil. Prognosis prediction was validated in external datasets. The expression of CTLA4, CAMK2N1, PLAU and CALML5 was enhanced in CAL-27 and SCC-25 cell lines, and CALML5 inhibited CAL-27 and SCC-25 cell viability. Conclusion: This study shares novel insights into HNSC classification and provides a reliable PCD-related prognostic signature for prognosis prediction and treatment for patients with HNSC.

A synthetic derivative of bioactive constituents from Isatis indigotica ameliorates hypersensitivity and arthritis by inhibiting JAK2-STAT3 pathway in mice.

The JAK-STAT pathway plays a crucial role in the signaling cascade associated with various cytokines that have been implicated in the pathogenesis of inflammatory diseases and myeloproliferative neoplasms (MPN). Among the isoforms of JAKs, the JAK2 subtype is primarily responsible for the function of hematopoietic system cells, making it a significant target in the treatment of MPN. However, the precise regulatory role of JAK2 in inflammatory diseases requires further investigation and confirmation. The current study employed a selective JAK2 inhibitor, ZT55, derived from Isatis indigotica roots, to examine its regulatory effects on inflammatory and immune responses in delayed-type hypersensitivity (DTH) and arthritis in mice. To evaluate the efficacy of ZT55 treatment, DNFB-induced DTH and collagen-induced arthritis (CIA) mouse models were utilized. T cells were cultured and subsequently analyzed for proliferation and activation using flow cytometry and EdU assay. Additionally, the maturation and function of dendritic cells were assessed through flow cytometry and ELISA. Our findings indicate that ZT55 significantly reduced DNFB-induced DTH and attenuated inflammation, cartilage degradation, and bone destruction in CIA mice. Moreover, ZT55 was found to inhibit the proliferation and activation of T cells and the maturation of dendritic cells by regulating the JAK2-STAT3 signaling pathway. These results suggest that selectively targeting the JAK2 isoform could have anti-inflammatory and immunosuppressive effects by regulating the adaptive and innate immune responses via the JAK2-STAT3 signaling pathway. Therefore, ZT55 has the potential to be a promising pharmaceutical candidate for the treatment of inflammatory and autoimmune diseases.

[Clinical Features and Outcomes of the Patients with B-Cell Chronic Lymphoproliferative Disease in the New Drug Era].

OBJECTIVE: To analyze the clinical characteristics of the patients with B-cell chronic lymphoproliferative disease（B-CLPD） in the new drug era and the effect of new drug treatment on efficacy and survival. METHODS: The clinical and laboratory data of 200 cases B-CLPD patients diagnosed between April 2015 and August 2021 were analyzed retrospectively. The clinical efficacy and survival of the patients under different treatments including Bruton tyrosine kinase（BTK） inhibitors， rituximab， and chemotherapy alone were analyzed. The prognostic factors affecting the survival of patients were analyzed by univarite analysis and multivariate analysis. RESULTS: There were 119 male（59.5％） and 81 female（40.5%） in 200 cases B-CLPD patients， the sex ratio(male/female) was 1.5∶1 with median age of 61（30- 91） years old. The distribution of subtypes were as fallows: 51 cases (25.5%) of chronic lymphocytic leukemia/small lymphocytic lymphoma（CLL/SLL）， 64（32.0%） cases of follicular lymphoma（FL）， 40（20.0%） cases mantle cell lymphoma（MCL）， 30（15.0%） cases of marginal zone lymphoma（MZL）， 10（5%） cases of lymphoplasmacytic lymphoma/waldenstrom macroglobulinemia（LPL/WM）， 5（2.5%） cases of B cell chronic lymphoproliferative disorders unclassified（B-CLPD-U） . The main clinical manifestation of 102 patients was lymph node enlargement, 32 cases were complicated with B symptoms. Among CLL/SLL patients， there were 12（23.5%） cases in Binet A and 39（76.5%） cases in Binet B/C. There were 29 patients（20.9%） in Ann Arbor or Lugano stage I-II and 110 cases（79.1%） in stage III-IV of other subtypes. The complete remission（CR） rate was 43.1%（25/58）， 40.2%（39/97）， 7.1%（1/14）， and overaIl response rate（ORR） was 87.9%（51/58）， 62.9%（61/97）， 28.6%（4/14） in the groups of BTK inhibitors， rituximab-based therapy， and chemotherapy alone. The 3-year OS rate and PFS rate in all patients was 79.2% and 72.4% respectively. The 3-year OS rate of patient with MZL, CLL/SLL, FL,WM was 94.7%, 87.7%, 86.8% and 83.3% respectively, while the 3-year OS rate of MCL was only 40.6%, which was significantly lower than other subtypes. The median OS of patients treated with BTK inhibitors and rituximab-based therapy was 20.5 and 18.5 months respectively， and the 3-year OS rate was 97.4% and 90.7%. However， the median PFS of patients receiving chemotherapy alone was 4 months， and the 1-year OS rate was 52.7%， which was statistically significant compared with the other two groups（P<0.05）. Univarite analysis showed that anemia， elevated lactate dehydrogenase， elevated ß2-microglobulin， and splenomegaly were the poor prognostic factors for OS（P<0.05）， elevated lactate dehydrogenase was also poor prognostic factors for PFS（P<0.05）. Multifactor analysis showed that anemia and elevated lactate dehydrogenase were the independent poor prognostic factors for survival（P<0.05）. CONCLUSION: The clinical features of B-CLPD was various， anemia and elevated lactate dehydrogenase are the prognostic factors for poor survival. BTK inhibitors and new immunotherapy can improve the survival and prognosis of patients in the new drug era.

Effects of high-intensity gymnastics on skeletal muscle proteins / Efeitos da ginástica de alta intensidade sobre as proteínas musculares esqueléticas / Efectos de la gimnasia de alta intensidad en las proteínas musculares esqueléticas

ABSTRACT Introduction: Although the current method of muscle stretching in gymnastics teaching in colleges and universities can reduce sports fatigue, it has been shown to have little effect on the well-being of athletes because it requires a long recovery time from psychological fatigue. Progressive muscle relaxation training is a method that uses the basic principle of sympathetic nerve activity to reduce the impact of negative emotions psychologically and relieve fatigue physiologically, requiring a further study of its impact on muscle protein. Objective: Explore the effect of high-intensity gymnastics on skeletal muscle protein and study the progressive muscle relaxation training method post-workout adjustment. Methods: After three weeks of training, excluding the standard deviations in the experimental data caused by the athletes' irregular movements, the athletes' blood lactate content and heart rate were counted and recorded. The collected data were analyzed using Excel software to integrate and compare the data using the T-test method. Results: After exercise training, the skeletal muscle function indices of the subjects increased to different degrees. From the point of view of heart rate recovery efficiency, the rate of heart rate decline of progressive relaxation training was higher than that of the two groups, and the degree of fluctuation was lower than that of the two groups, indicating that the level of recovery in heart rate of progressive relaxation training was better. Conclusion: The action of the high-intensity gymnastics team has a good effect on improving the athletes' skeletal muscle and skeletal muscle proteins. Post-exercise conditioning training plays an important role in athletes' physical recovery. Level of evidence II; Therapeutic studies - investigation of treatment outcomes.

RESUMO Introdução: Embora o método de alongamento muscular atual no ensino de ginástica em faculdades e universidades consiga reduzir a fadiga esportiva, tem se mostrado pouco eficaz no bem-estar dos atletas por exigir grande tempo de recuperação da fadiga psicológica. O treinamento progressivo de relaxamento muscular é um método que usa o princípio básico da atividade nervosa simpática para reduzir o impacto das emoções negativas psicologicamente e aliviar a fadiga fisiologicamente, necessitando de mais estudos do seu impacto sobre a proteína muscular. Objetivo: Explorar o efeito da ginástica de alta intensidade sobre as proteínas musculares esqueléticas e estudar o método de treinamento progressivo de relaxamento muscular no ajuste pós-treino. Métodos: Após 3 semanas de treinamento, excluídos os desvios-padrão nos dados experimentais causados pelos movimentos irregulares dos atletas, foram contabilizados e registrados os conteúdos de lactato sanguíneo e frequência cardíaca dos atletas. Analisou-se os dados coletados, com o software Excel, para integrar e comparar os dados pelo método de teste-T. Resultados: Após o treinamento do exercício, os índices de função muscular esquelética dos sujeitos aumentaram em diferentes graus. Do ponto de vista da eficiência da recuperação da frequência cardíaca, a taxa de declínio da frequência cardíaca do treinamento de relaxamento progressivo foi maior do que a dos dois grupos, o grau de flutuação foi menor do que o dos dois grupos, indicando que o nível de recuperação na frequência cardíaca do treinamento de relaxamento progressivo foi melhor. Conclusão: A ação da equipe de ginástica de alta intensidade tem um bom efeito na melhoria do músculo esquelético e das proteínas musculares esqueléticas dos atletas. O treinamento de condicionamento pós-exercício desempenha um papel importante na recuperação física dos atletas. Nível de evidência II; Estudos terapêuticos - investigação dos resultados do tratamento.

RESUMEN Introducción: Aunque el método actual de elongación muscular en la enseñanza de la gimnasia en colegios y universidades consigue reducir la fatiga deportiva, se ha demostrado que tiene poco efecto en el bienestar de los atletas porque requiere un largo tiempo de recuperación de la fatiga psicológica. El entrenamiento de la relajación muscular progresiva es un método que utiliza el principio básico de la actividad nerviosa simpática para reducir el impacto de las emociones negativas desde el punto de vista psicológico y aliviar la fatiga desde el punto de vista fisiológico, lo que requiere un estudio más profundo de su impacto en la proteína muscular. Objetivo: Explorar el efecto de la gimnasia de alta intensidad sobre la proteína del músculo esquelético y estudiar el método de entrenamiento de relajación muscular progresiva en el ajuste posterior al entrenamiento. Métodos: Después de 3 semanas de entrenamiento, excluyendo las desviaciones estándar en los datos experimentales causadas por los movimientos irregulares de los atletas, se contó y registró el contenido de lactato en sangre y la frecuencia cardíaca de los atletas. Los datos recogidos se analizaron, con el programa informático Excel, para integrar y comparar los datos mediante el método de la prueba T. Resultados: Tras el entrenamiento con ejercicios, los índices de función del músculo esquelético de los sujetos aumentaron en diferentes grados. Desde el punto de vista de la eficacia de la recuperación de la frecuencia cardíaca, el índice de disminución de la frecuencia cardíaca del entrenamiento de relajación progresiva fue mayor que el de los dos grupos, el grado de fluctuación fue menor que el de los dos grupos, lo que indica que el nivel de recuperación de la frecuencia cardíaca del entrenamiento de relajación progresiva fue mejor. Conclusión: La acción del equipo de gimnasia de alta intensidad tiene un buen efecto en la mejora del músculo esquelético y de las proteínas del músculo esquelético de los atletas. El entrenamiento de acondicionamiento posterior al ejercicio desempeña un papel importante en la recuperación física de los deportistas. Nivel de evidencia II; Estudios terapéuticos - investigación de los resultados del tratamiento.

[Effect of miR-203/CREB1 Signaling Regulation Mediated by DNA Methylation on the Proliferation and Apoptosis of Multiple Myeloma Cells].

OBJECTIVE: To investigate the effect of miR-203/CREB1 signaling regulation mediated by DNA methylation on the proliferation, invasion and apoptosis of multiple myeloma (MM) cells. METHODS: The methylation level of miR-203 in the RPMI 8226 cells was detected by bisulfite sequcucing polymerase chain reaction (BSP). The mRNA expression of miR-203 was measured by quantitative real-time polymerase chain reaction. RPMI 8226 cells were treated with DNA methyltransferase inhibitor 5-Aza-2'-deoxycytidine (5-Aza-CdR). The miR-203 mimic in MM cell line RPMI 8226 was transfected to establish overexpressed miR-203 cell. The proliferation, invasion ability and apoptosis of RPMI 8226 cell was detected by CCK-8 assay, Transwell, and flow cytometry, respectively. The targeting relationship between miR-203 and CREB1 was verified by double luciferase report assay. Western blot was used to detect the expression of CREB1 protein. RESULTS: Hypermethylation of miR-203 promoter region and low expression level of miR-203 mRNA were detected in the RPMI 8226 cells, which showed that demethylation could induce the expression of miR-203. The proliferation and invasion ability of RPMI 8226 cells after treated by 5-Aza-CdR were inhibited, and showed statistical significance as compared with blank control group (both P<0.05)，while the apoptosis rate was promoted (P<0.05). The proliferation, invasion ability and apoptosis of overexpressed miR-203 were the same as the demethylation group. Double luciferase report assay confirmed that CREB1 was the direct target of miR-203. The protein level of CREB1 was inhibited by demethylation and showed statistical significance as compared with control group (P<0.05). CONCLUSION: MiR-203 targeting CREB1 mediated by DNA methylation leads to maintain the malignant biological behaviors of MM cells.

Simultaneous determination of YZG-331 and its metabolites in monkey blood by liquid chromatography-tandem mass spectrometry.

N6-[(S)-1- (phenyl)-propyl]-adenine riboside (YZG-331) is being developed as a novel sedative and hypnotic agent. The hydroxylated metabolites of YZG-331 have the same mass transition ion pair, making their determination in blood challenging. In this study, a rapid and sensitive liquid chromatography-tandem mass spectrometry method was developed for the simultaneous determination of YZG-331 and its metabolites M1 (hydrolysis), M2 and M4 (hydrolysis and hydroxylation), M3, M5 and M6 (hydroxylation) in monkey blood. Propranolol was used as the internal standard (IS). Blood samples were prepared using a simple protein precipitation with acetonitrile. The chromatographic separation was performed on an Eclipse Plus C18 column (2.1 × 50 mm, 3.5 µm) at a flow rate of 0.3 mL/min with a gradient mobile phase of methanol/water containing 0.5 % formic acid (v/v). Detection was carried out on a triple quadrupole mass spectrometer in positive ion multiple reaction monitoring mode. The optimized mass transition ion pairs for quantitation were 386→254 for YZG-331, 254→136 for M1, 270→136 for M2 and M4, 402→136 for M3, M5 and M6 and 260→183 for IS. Acceptable linearity was obtained for the analytes over the range of 15-2000 ng/mL for YZG-331, 3-400 ng/mL for M1-M6. The lower limits of the quantification were 15 ng/mL for YZG-331, 3 ng/mL for M1-M6. The intra- and inter-day precisions wre within 10.5 % for all analytes, while the accuracy ranged from -8.3 %-8.8 %. There was no obvious matrix effect and the recoveries of the analytes were 90.6 %-118.2 %. The analytes were proved to be stable during all sample storage, preparation and analytic procedures. The sensitive and rapid LC-MS/MS method for YZG-331 in monkey blood has been applied to pharmacokinetic studies of YZG-331 in monkeys. The oral bioavailability of YZG-331 in monkeys is 74.1 %.

Discovery and evaluation of ZT55, a novel highly-selective tyrosine kinase inhibitor of JAK2V617F against myeloproliferative neoplasms.

BACKGROUND: The JAK2-STAT signaling pathway plays a critical role in myeloproliferative neoplasms (MPN). An activating mutation in JAK2 (V617F) is present in ~ 95% of polycythemia vera, essential thrombocythemia, and primary myelofibrosis cases. This study aims to explore the selective JAK2V617F inhibitor, evaluate the efficacy and possible mechanism of ZT55 on MPN. METHODS: HTRF assays were conducted to evaluate the selective inhibition of ZT55 for JAKs. Cell apoptosis, proliferation, and cycle arrest assays were performed to examine the effect of ZT55 on HEL cell line with JAK2V617F mutation in vitro. Western analysis was used to monitor the expression and activity of proteins on JAK2/STAT pathway. A mice xenograft model was established to evaluate the antitumor efficacy of ZT55 in vivo. Peripheral blood samples from patients with the JAK2V617F mutation were collected to estimate the effect of ZT55 on erythroid colony formation by colony-forming assay. RESULTS: We found that ZT55 showed a selective inhibition of a 0.031 µM IC50 value against JAK2. It exhibited potent effects on the cellular JAK-STAT pathway, inhibiting tyrosine phosphorylation in JAK2V617F and downstream STAT3/5 transcription factors. ZT55 inhibited the proliferation of the JAK2V617F-expressing HEL cell line, leading to cell cycle arrest at the G2/M phase and induction of caspase-dependent apoptosis. Notably, ZT55 also significantly suppressed the growth of HEL xenograft tumors in vivo. Further evaluation indicated that ZT55 blocked erythroid colony formation of peripheral blood hematopoietic progenitors from patients carrying the JAK2V617F mutation. CONCLUSION: These results suggest that ZT55 is a highly-selective JAK2 inhibitor that can induce apoptosis of human erythroleukemia cells by inhibiting the JAK2-STAT signaling.

[Effects of Triptolide on MMP-9 Expression and Inducing Apoptosis of Multiple Myeloma Cells by Inhibiting SMYD3].

OBJECTIVE: To investigate the effect of triptolide(TPL) on proliferation and apoptosis of RPMI8226 cells and its mechanism. METHODS: MTT assay was used to measure the proliferation of RPMI8226 cells after treatment with different concentration (10, 20, 40, 80 and 160 nmol/L) of TPL for different incubation time (24 h, 48 h and 72 h). The cell apoptosis was detected by flow cytometry, the mRNA expressions of SMYD3 and MMP-9 were measured by quantitative real-time PCR, the protein level of H3K4me2 and H3K4me3 in RPMI8226 cells was assayed by Western blot. RESULTS: TPL inhibited RPMI8226 cell proliferation, and the inhibitory rate of cell proliferation increased significantly in a dose- and time-dependent manner(P<0.05), the RPMI8226 cell apoptosis was induced by treatment with 40, 80 and 160 nmol/L TPL (P<0.05), the qRT-PCR showed that treatment of RPMI8226 cells with TPL down-regulated the mRNA expression of SMYD3 in a dose-dependent manner(P<0.05). Compared with the blank group, the mRNA expression level of MMP-9 in RPMI8226 cells transfected by siRNA-SMYD3 was significantly depressed. Western blot showed that the protein levels of H3K4me2 and H3K4me3 were decreased in a dose-dependent manner after TPL treatment(P<0.05). Compared with the blank group and siRNA negative group, the protein level of H3K4me2 and H3K4me3 in RPMI8226 cells transfected by siRNA-SMYD3 also were significantly depressed(P<0.05). CONCLUSION: TPL can significantly inhibit the proliferation of RPMI8226 cells and induce their apoptosis, which may be related to the inhibition of SMYD3 expression by TPL- down-regulating the H3K4 methylation and the activating the MMP-9 transcription.

[Effects of triptolide on the apoptosis and H3K4 protein methylation in multiple myeloma cells].

OBJECTIVE: To investigate the effects of triptolide (Chinese Traditional Medicine)on the apoptosis and H3K4 protein methylation in multiple myeloma cells. METHODS: The RPMI8226 cells were cultured with different concentrations(10,20,40,80 and 160 nmol/L)of triptolide for different incubation time (24 h,48 h and 72 h). The inhibition of triptolide on RPMI8226 cell proliferation was detected by MTT assay. Apoptosis and cell cycle distribution were evaluated by flow cytometry.The expressions of H3K4me2 and trimethylation of histone H3 lysine 4(H3K4me3) in RPMI8226 cells were assayed by Western blot. The changes of expressions of histone methylase SET and MYND domain containing 3(SMYD3) and histone demethylase lysine specific demethylase 1(LSD1) in RPMI8226 cells were verified by qRT-PCR. RESULTS: Triptolide had obvious inhibitive effects on proliferation of RPMI8226 cells and showed a dose-and time-dependent manner(P<0.05). Triptolide induced apoptosis and G2/M cell cycle arrest in a dose-dependent manner(P<0.05). Triptolide decreased histone H3K4me2 and H3K4me3 expression in a dose-dependent manner(P<0.05, P<0.01). SMYD3 was significantly depressed at protein expression in a dose-dependent manner(P<0.05), but LSD1 was up-regulated (P<0.05). CONCLUSIONS: Triptolide could inhibit RPMI8226 cell proliferation,induce the apoptosis and cause G2/M arrest,meanwhile,significantly inhibit the protein expressions of H3K4me2 and H3K4me2 with alter the expression of SMYD3 and LSD1.The effects is probably related to the antitumor mechanism of MM cells induced by triptolide.

[Lignanoids from an aqueous extract of the roots of Codonopsis pilosula].

Sixteen lignanoids were isolated from an aqueous extract of the commonly used Chinese traditional medicine Dangshen, the dried roots of Codonopsis pilosula, by using a combination of various chromatographic techniques, including silica gel, macroporous adsorbent resin, MCI resin, sephadex LH-20, and reversed phase semi-preparative HPLC. On the basis of spectral data analysis, their structures were elucidated and identified as（-）-（7R,7'R,8R,8'S）-4,4'-dihydroxy-3,3',5,5',7-pentamethoxy-2,7'-cyclolignane（1）,（-）-（7R,8S）- dihydrodehydrodiconiferyl alcohol 4-O-ß-D-glucopyranosyl-（1'''→2''）-ß-D-glucopyranoside（2）,（-）-（7R,8S）- dihydrodehydrodiconiferyl alcohol（3）,（+）-（7S,8R）-dehydrodiconiferyl alcohol（4）,（+）-balanophonin（5）,（+）- demethoxypinoresinol（6）,（+）-pinoresinol（7）,（+）-epipinoresinol（8）,（-）-syringaresinol（9）,（-）-medioresinol（10）,（-）-lariciresinol（11）,（-）-secoisolariciresinol（12）,（-）-ent-isolariciresinol（13）,（+）-（7S,8S）-3-methoxy-3',7- expoxy-8,4'-neolignan-4,9,9'-triol（14）,（+）-（7S,8R）-3',4-dihydroxy-3-methoxy-8,4'-neolignan（15）, and（-）-（7R,8R）-3',4-dihydroxy-3-methoxy-8,4'-neolignan（16）. All these compounds were isolated from C. pilosula for the first time, while compound 1 is a new natural product of 2,7'-cyclolignan and 2 is a new 4',7-epoxy- 8,3'-neolignan diglucoside. Compound 12 showed activity against Fe(2+)-cysteine induced rat liver microsomal lipid peroxidation with an inhibition ratio of（63.4 ± 8.3） % at 1×10(-5) mol·L(-1).

4-Hydroxybenzyl-substituted amino acid derivatives from Gastrodia elata.

Seven new 4-hydroxybenzyl-substituted amino acid derivatives (1-7), together with 11 known compounds, were isolated from an aqueous extract of the rhizomes of Gastrodia elata Blume. Their structures were determined by spectroscopic and chemical methods. Compounds 1-3 are pyroglutamate derivatives containing 4-hydroxybenzyl units at the N atom and 4-7 are the first examples of natural products with the 4-hydroxybenzyl unit linked via a thioether bond to 2-hydroxy-3-mercaptopropanoic acid (4-6) and 2-hydroxy-4-mercaptobutanoic acid (7), which would be biogenetically derived from cysteine and homocysteine, respectively. The structures of 1 and 2 were verified by synthesis, while the absolute configurations of 4, 5 and 7 were assigned using Mosher's method based on the MPA determination rule of Δδ RS values. The known compound 4-(hydroxymethyl)-5-nitrobenzene-1,2-diol (8) exhibited activity against Fe(2+)-cysteine induced rat liver microsomal lipid peroxidation with IC50 values of 9.99×10(-6) mol/L.

Methylation of secreted frizzled related protein gene in acute leukemia patients in China.

BACKGROUND: DNA methylation of CpG islands within the promoters of specific genes may play roles in tumor initiation and progression. It has been suggested such events may serve as critical check points. METHODS: The present study analyzed the methylation status of CpG islands within the promoters of secreted frizzled-related proteins (SFRPs) in 87 acute leukemia (AL) patients, 20 normal controls, and four AL cell lines. 5-aza-2'- deoxycytidine (5-Aza-CdR), an inhibitor of DNA methylation, was employed to determine its effect on SFRP expression. RESULT: Methylation of at least one SFRP promoter was observed in 69% of the AL patients analyzed. In addition, methylation of all four SFRP promoters was observed in Molt-4, Jurkat, HL60 and NB4 cells. In Jurkat cells, methylation levels of four SFRP promoters decreased in a dose-dependent manner upon treatment with 5-Aza-CdR, which coincided with increased mRNA expression. With increasing 5-Aza-CdR concentrations, the expression of DNA methyltransferases, DNMT3A and DNMT3B, significantly decreased in a dose-dependent manner. CONCLUSION: The present study demonstrated that SFRP gene methylation may be involved in AL progression, with a possible epigenetic mechanism influencing Wnt signaling.

[The significance of methylation status of secreted frizzled-related protein gene promoter in acute leukemia].

OBJECTIVE: To investigate the relationship between promoter hypermethylation of secreted frizzled-related protein (SFRP) gene and acute leukemia (AL). METHODS: We examined the promoter methylation status of SFRP1, 2, 4 and 5 in primary or relapsed AL patients, cell lines (HL60, NB4, Molt-4 and Jurkat) and peripheral blood mononuclear cells from healthy people with methylation-specific PCR (MSP). RESULTS: None of the normal mononuclear cells showed methylation of any SFRP genes. The frequencies of aberrant methylation among the samples were 33.9% (20/59) for SFRP1, 23.7% (14/59) for SFRP2, 6.8% (4/59) for SFRP4 and 10.2% (6/59) for SFRP5 in acute myelocytic leukemia (AML), and 39.3% (11/28) for SFRP1, 28.6% (8/28) for SFRP2, 25.0% (7/28) for SFRP4 and 32.1% (9/28) for SFRP5 in acute lymphoblastic leukemia (ALL). Hypermethylation of SFRP1, 2, 5 genes were present in the 4 AL cell lines. SFRP4 was methylated in NB4, Molt-4 and Jurkat cell lines. However, methylation and unmethylation of SFRP4 were both detected in HL60. CONCLUSIONS: Hypermethylation of SFRP genes is a common event in the evolution of AL. Methylation of SFRP genes might serve as potential independent biomarkers for early detection of AL.

[Inhibitory effect of VPA on multiple myeloma U266 cell proliferation and regulation of histone acetylation].

This study was aimed to investigate the effects of sodium valproate (VPA) on the proliferation and regulation of histone acetylation of multiple myeloma cell line U266. U266 cells were treated with VPA. Cell proliferation was determined by CCK-8 assay, and cell cycle were analyzed by flow cytometry (FCM). The expression level of HDAC1 mRNA was detected by RT-PCR, and the protein levels of HDAC1 and histone H3, H4 acetylation was detected by Western blot. The results showed that the VPA inhibited the proliferation of U266 cells in concentration-and time-dependent manners.After exposure to different concentrations of VPA for 48 hours, the proportion of G(0)/G(1) cells increased, while the proportion of S phase cells decreased. The cell cycle was arrested obviously in G(0)/G(1) phase (p < 0.05). The expression of HDAC1 mRNA was inhibited, and the protein level of HDAC1 was down-regulated, while the histone H3/H4 acetylation was up-regulated in U266 cells. It is concluded that the VPA can inhibit cell proliferation of U266 and induce G(0)/G(1) phase arrest. The increase of histone H3/H4 acetylation resulting from inhibiting HDAC1 by VPA might be considered as a possible mechanism.

[Methylation status of CpG islands in secreted frizzled-related protein gene promoter region of malignant hematopoietic cell lines].

To investigate the methylation status of CpG islands of the secreted frizzled-related protein (SFRP) gene promoter region in malignant hematopoietic cell lines, and to explore the possible relationship of CpG abnormal methylation status with pathogenic mechanism of hematologic malignancies. Methylation-specific PCR was used to detect the status of SFRP gene promoter region in nine malignant hematopoietic cell lines and peripheral blood mononuclear cells from healthy people. The results indicated that hypermethylation of 2 genes coding for SFRP1 and 2 were present in nine malignant hematopoietic cell lines, however, methylation and unmethylation of SFRP4 were both detected in CA46, HL60 and U937 cell lines, and SFRP5 in U266 as well. None of the normal mononuclear cells showed methylation of SFRP 1-5 genes. It is concluded that the hypermethylation of SFRP genes is related to the evolution of malignant hematopoiesis. Methylation of SFRP genes may serve as potential independent biomarkers for early detection of hematologic malignancies.