A randomized controlled trial of indobufen versus aspirin in the prevention of bridging restenosis after coronary artery bypass grafting / 中华心血管病杂志

Objective: To compare the efficacy and safety between indobufen and aspirin in the prevention of restenosis of bridge vessels at 1 year after off-pump coronary artery bypass grafting. Methods: This study was a prospective cohort study. We selected 152 patients who received coronary artery bypass grafting in Beijing Anzhen Hospital from December 2016 to December 2018. Patients were divided into the indobufen group and the aspirin group. Patients in the aspirin group were treated with aspirin and clopidogrel, and patients in the indobufen group were treated with indobufen and clopidogrel. During the 1-year follow-up, the rate of restenosis of saphenous vein bridge and internal mammary artery bridge, the rate of adverse cardiac events and adverse reactions were compared between the two groups. The levels of fibrinogen (FIB), D-dimer (D-D), thrombomodulin (TM) and thrombin-activatable fibrinolysis inhibitor (TAFI) were compared before and after antiplatelet therapy. Results: There were 76 cases in the indobufen group, including 57 males (75.0%), aged (60.3±6.6) years. There were 76 cases in the aspirin group, including 62 males (81.6%), aged (59.7±7.2) years. Baseline data were comparable between the two groups (P>0.05). During the follow-up, 3 cases were lost to follow up. Follow-up was completed in 74 patients in the indobufen group and 75 in the aspirin group. A total of 268 bridging vessels were grafted in the indobufen group and 272 in the aspirin group. One year after surgery, the patency rates of great saphenous vein bridge and internal mammary artery bridge were 94.5% (189/200) and 97.1% (66/68) in the indobuphen group, and 91.3% (189/207) and 96.9% (63/65) in the aspirin group, respectively. There was no significant difference in patency rate of great saphenous vein bridge and internal mammary artery bridge between the two groups (χ²=0.282, 0.345, P>0.05). The total incidence of adverse cardiac events was 5.4% (4/74) in the indobufen group and 6.7% (5/75) in the aspirin group (χ²=0.126, P>0.05). The overall incidence of gastrointestinal adverse reactions was significantly lower in the indobufen group than in the aspirin group (4.1% (3/74) vs. 13.3% (10/75), χ²=4.547, P<0.05). The levels of FIB, D-D, TM and TAFI in the two groups were lower than those before surgery (P<0.05), and there was no statistical significance between the two groups at baseline and post-operation (P>0.05). Conclusion: The efficacy of indobufen combined with clopidogrel in the prevention of 1-year restenosis after coronary artery bypass graft is similar to that of aspirin combined with clopidogrel, but the incidence of adverse reactions is lower, and the safety is higher in patients treated with indobufen combined with clopidogrel compared to aspirin combined with clopidogrel strategy.

Sodium 4-phenylbutyrate Attenuates High-fat Diet-induced Impaired Spermatogenesis / 生物医学与环境科学（英文）

OBJECTIVE@#To determine the mitigating effects of sodium 4-phenylbutyrate (4-PBA) on high-fat diet (HFD)-induced spermatogenesis dysfunction.@*METHODS@#Male rats (n = 30) were randomly divided into three groups: control, HFD, and 4-PBA (HFD +4-PBA). After 13 weeks, rats were euthanized. Testes and epididymis were harvested for further analysis. Sex hormones were detected, and hematoxylin and eosin staining was performed to examine the histological changes in the testes. Semen samples were collected to evaluate sperm quality. Spermatogenic cell apoptosis was detected by TUNEL assay.@*RESULTS@#Compared with the control group, the final body weight and body weight gain were significantly higher in HFD-fed rats, while the testicle/body weight ratios were lower (P < 0.05). In HFD-fed rats, obvious pathological changes in the testicular tissue were observed. Treatment with 4-PBA attenuated HFD-induced histological damage, ameliorated the HFD-induced decrease in serum testosterone (T), and reduced the rate of testicular cell apoptosis (P < 0.05) in obese male rats. Finally, 4-PBA significantly improved semen parameters in HFD rats (P < 0.05).@*CONCLUSION@#HFD exposure induced detrimental effects on spermatogenesis, semen quality, serum T level, and testicular cell apoptosis in rats. Treatment with 4-PBA ameliorated HFD？induced impaired spermatogenesis via inhibition of apop-tosis in rats. 4-PBA may have therapeutic value in the treatment of obesity？related impairment of spermatogenesis.

The F309S mutation increases factor VIII secretion in human cell line

OBJECTIVES: The capacity of a human cell line to secrete recombinant factor VIII with a F309S point mutation was investigated, as was the effect of the addition of chemical chaperones (betaine and sodium-4-phenylbutyrate) on the secretion of factor VIII. METHODS: This work used a vector with a F309S mutation in the A1 domain to investigate FVIII production in the HEK 293 human cell line. Factor VIII activity was measured by chromogenic assay. Furthermore, the effects of chemical drugs on the culture were evaluated. RESULTS: The addition of the F309S mutation to a previously described FVIII variant increased FVIII secretion by 4.5 fold. Moreover, the addition of betaine or sodium-4-phenylbutyrate increased the secretion rate of FVIIIÄB proteins in HEK 293 cells, but the same effect was not seen for FVIIIÄB-F309S indicating that all the recombinant protein produced had been efficiently secreted. CONCLUSION: Bioengineering factor VIII expressed in human cells may lead to an efficient production of recombinant factor VIII and contribute toward low-cost coagulation factor replacement therapy for hemophilia A. FVIII-F309S produced in human cells can be effective in vivo.

Synthesis and biological evaluation of a mutual prodrug of norfloxacin and fenbufen

Objectives: The study aimed to synthesize a mutual prodrug of norfloxacin and fenbufen with an objective of obtaining an effective and safer anti-inflammatory drug with useful antimicrobial actions

Methods: An amide-based mutual prodrug [NF-FN] was prepared following a single-step synthesis by condensing norfloxacin with fenbufen under appropriate laboratory conditions. Its structure was established on the basis of IR, NMR, Mass spectral data and elemental analysis. The prodrug [NF-FN] was evaluated for in-vitro antibacterial activity against two grampositive [Staphylococcus aureusand Bacillus subtilis] and two gram negative bacterial strains [Escherichia coli and Klebsiella pneumonia]. The in-vivo antiinflammatory activity and ulcerogenicity of the synthesized prodrug were investigated in Wistar albino rats at the doses of 10 and 30 mg/kg body weight, respectively

Results: The synthesized prodrug [NF-FN] showed very good activity against S. aureus and E. coli with MIC-6.25 mg/ mL, and good activity against B. subtilis and K. pneumonia with MIC-12.5 mg/mL. Its anti-inflammatory activity was found to be better than that of the parent drug fenbufen. It was also observed to less severe on gastric mucosa in comparison to reference drug, fenbufen

Conclusion: The prodrug showed promising results as anti-inflammatory agent however, its antibacterial action was found to be slightly weaker than the other parent drug norfloxacin

Advanced glycated albumin induces macrophage apoptosis via activating caspase-12 pathway / 生理学报

The purpose of the present study was to investigate the effect of advanced glycated albumin (AGE-alb) on the activation of caspase-12, a key molecule in endoplasmic reticulum stress (ERS)-associated apoptotic pathway, and to elucidate the underlying molecular mechanisms of macrophage apoptosis. RAW264.7 macrophages were treated with AGE-alb (2, 4 and 6 g/L), control albumin (C-alb, 4 g/L), tunicamycin (TM, 4 mg/L), or pretreated with 4-phenylbutyric acid (PBA, 5 mmol/L) for 1 h and then treated with AGE-alb (4 g/L). After incubation for 24 h, the cell viability and apoptosis were determined by using MTT assay and TUNEL detection kit, respectively. Lactate dehydrogenase (LDH) activity in media was determined by using an assay kit. The protein levels of caspase-12 were examined by Western blot analysis. The results showed that like TM (an ERS inducer), incubation with AGE-alb led to significant decrease in viability and increase in LDH activity in media and apoptotic rate in a dose-dependent manner. In addition, AGE-alb induced activation of caspase-12 especially at the concentration of 4 and 6 g/L (P < 0.01), which was similar to TM. However, PBA (an ERS inhibitor) protected RAW264.7 macrophages from AGE-alb-induced decrease in viability and increases in LDH activity and apoptosis. Moreover, PBA also inhibited the caspase-12 activation induced by AGE-alb (P < 0.05). These results suggest that AGE-alb may induce apoptosis in RAW 264.7 macrophages, and the mechanism may be related to the activation of ERS-associated apoptotic pathway mediated by caspase-12.

Homocysteine activates T cells by enhancing endoplasmic reticulum-mitochondria coupling and increasing mitochondrial respiration

Hyperhomocysteinemia (HHcy) accelerates atherosclerosis by increasing proliferation and stimulating cytokine secretion in T cells. However, whether homocysteine (Hcy)-mediated T cell activation is associated with metabolic reprogramming is unclear. Here, our in vivo and in vitro studies showed that Hcy-stimulated splenic T-cell activation in mice was accompanied by increased levels of mitochondrial reactive oxygen species (ROS) and calcium, mitochondrial mass and respiration. Inhibiting mitochondrial ROS production and calcium signals or blocking mitochondrial respiration largely blunted Hcy-induced T-cell interferon γ (IFN-γ) secretion and proliferation. Hcy also enhanced endoplasmic reticulum (ER) stress in T cells, and inhibition of ER stress with 4-phenylbutyric acid blocked Hcy-induced T-cell activation. Mechanistically, Hcy increased ER-mitochondria coupling, and uncoupling ER-mitochondria by the microtubule inhibitor nocodazole attenuated Hcy-stimulated mitochondrial reprogramming, IFN-γ secretion and proliferation in T cells, suggesting that juxtaposition of ER and mitochondria is required for Hcy-promoted mitochondrial function and T-cell activation. In conclusion, Hcy promotes T-cell activation by increasing ER-mitochondria coupling and regulating metabolic reprogramming.

The role of cell apoptosis mediated by endoplasmic reticulum stress (ERS) of deep tissue injury of pressure ulcer of rats / 中国应用生理学杂志

<p><b>OBJECTIVE</b>To observe the the expression of endoplasmic reticulum stress (ERS) related factors in deep tissue injury (DTI) at pressure ulcer rat and to investigate the ERS mechanism of DTI in muscle tissue and protective effect of 4-phenylbutyric acid (4-PBA) in local tissue.</p><p><b>METHODS</b>Fifty male SD rats were randomly devided into control group, model group, experimental group NS group and PBA group, the experimental groups were divided into 4 d, 7 d, 14 d and 21 d group according to the observation time (n = 5). Rats in the PBA group were administrated with gastric perfusion of 4-PBA after the modeling; the NS group was given normal saline of the same quantity. Using HE staining to observe morphologic character. The expression of glucose regulated protein 78 (GRP78), CHOP, Caspase 12 were detected by immunohistochernical staining. Cell apoptosis was detected by TUNEL assay.</p><p><b>RESULTS</b>HE staining results showed that each group demonstrated compression injury compared with control group: cellular swelling, ompaction of nuclear, and apoptosis in muscle tissue. The new muscle fiber in 4-PBA group fused faster than those in NS group. The number of TUNEL positive cells peaked at 4 day after compression, then got decreased on day 7 in muscle tissue, apoptosis positive cells were diminished after 4-PBA treatment. The immunohistochemical staining results showed that the expression of protein GRP78, CHOP, Caspase 12 peakd 4 d after modeling and decreased gradually. The GRP78, CHOP, Caspase 12 protein expression were significantly higher than those of PBA group at all time points (P < 0.05).</p><p><b>CONCLUSION</b>Cell apoptosis induced by endoplasmic reticulum stress took part in deep tissue injury resulting of pressure ulcer, which mechanism might be related to reducing apoptosis mediated by CHOP, Caspase 12.</p>

Effects of 4-phenylbutyric acid on carbon tetrachloride-induced acute liver injury in mice / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To explore the effects and the molecular mechanisms of 4-phenylbutyric acid (PBA) on carbon tetraehloride (CCl4)-induced acute liver injury in mice.</p><p><b>METHODS</b>Sixty adult, healthy, male ICR mice were divided equally into the control group, PBA group, CCl4 12 h group, CCl4 24 h group, CCl4 48 h group, CCl4 72 h group, PBA+CCl4 12 h group, PBA+CCl4 24 h group, PBA+CCl4 48 h group, and PBA+CCl4 72 h group. The CCl4 groups and the PBA+CCl4 groups were intraperitoneally (i.p.) injected with CCl4 (300 mL/kg). In the PBA+CCl4 groups, the mice were i.p. injected with PBA (400 mg/kg). All mice were sacrificed to collect blood and liver specimens at different time points after the CCl4 treatment. Serum alanine aminotransferase (ALT) was detected. Histological examination was performed using hematoxylin-eosin staining and light microscopy, and apoptosis was detected using terminal transferase dUTP nick end labeling. The hepatic distribution of proliferating cell nuclear antigen (PCNA) was detected by immunohistochemistry. The hepatic protein expression of glucose-regulated protein (GRP78), C/EBP homologousprotein (CHOP), phosphorylated c-Jun N-terminal kinases (p-JNK), phosphorylated eukaryotic initiation factor 2a subunit (p-eIF2a), phosphorylated serine threonine kinase (p-akt), and nuclear factor-kappa B p65 (NF-kappa Bp65) were determined by western blot.</p><p><b>RESULTS</b>The serum ALT level in the PBA+CCl4 groups was reduced as compared with that in the CCl4 groups at the various time points examined.The liver-to-body weight ratio of two groups showed a significant difference only at the 48 h time point (P<0.01). PBA reduced the degree of hepatic necrosis and apoptosis caused by CCl4, and reduced the expression of hepatic GRP78 and other endoplasmic reticulum stress-related proteins (P<0.01). The protein levels of p-akt, NF-kappa Bp65 and PCNA was significantly decreased in the PBA+CCl4 groups (P<0.01).</p><p><b>CONCLUSION</b>The endoplasmic reticulum stress inhibitor PBA alleviated acute hepatic necrosis and apoptosis but restrained hepatic proliferation.</p>

Self-rated health in Brazilian adults and elderly: Data from the National Household Sample Survey 2008 / Percepción de la salud en adultos y ancianos brasileños: datos de la Encuesta Nacional de Hogares 2008

Objective. To investigate prevalence of poor self-rated health and its association with individual and household-level characteristics among adults and elderly in Brazil. Materials and methods. Cross-sectional study with Brazilian National Household Sample Survey 2008 (n=257 816). Crude and multilevel-adjusted Poisson regression models were fitted. Results. After adjusted analysis, poor self-rated health was significantly associated with higher household income, living alone, not having piped water nor garbage collection, lower education, not having health insurance, female sex, higher age, being a current or previous smoker, physical inactivity, having chronic diseases, having physical impairment. Subjects living in rural areas also had higher prevalence of poor self-rated health. The factors most strongly associated with the outcome were physical impairment and reporting three or more chronic diseases. Conclusions. Socioeconomic, health related behaviors, and physical health were associated with poor self-rated health.

Objetivo. Investigar la prevalencia de la percepción negativa de salud y su asociación con características individuales a nivel de los hogares en adultos y adultos mayores de Brasil. Material y métodos. Estudio transversal con datos de la Encuesta Nacional de Hogares de 2008 (n=257 816). Se estimaron modelos de regresión de Poisson multinivel crudos y ajustados. Resultados. Después del análisis ajustado, la autopercepción negativa de salud se asoció significativamente con mayor ingreso, vivir solo, no tener agua corriente ni recolección de basura, baja educación, carecer de seguro de salud, sexo femenino, mayor edad, tabaquismo, inactividad física, enfermedades crónicas y deterioro físico. Los habitantes de zonas rurales también tuvieron mayor prevalencia de percepción negativa. Los factores más fuertemente asociados fueron impedimento físico y presentación de tres o más enfermedades crónicas. Conclusiones. Factores socioeconómicos, comportamientos relacionados con la salud y salud física se asociaron con la percepción negativa.

Morphological and molecular identification of ticks infesting Boa constrictor (Squamata, Boidae) in Manaus (Central Brazilian Amazon) / Identificação morfológica e molecular de carrapatos coletados de jiboias (Boa constrictor) (Squamata, Boidae) da zona urbana de Manaus

The Boa constrictor is one of the world's largest vertebrate carnivores and is often found in urban areas in the city of Manaus, Brazil. The morphological identification of ticks collected from 27 snakes indicated the occurrence of Amblyomma dissimile Koch 1844 on all individuals sampled. In contrast, Amblyomma rotundatum Koch was found on only two snakes. An analysis of the 16S rRNA molecular marker confirmed the morphological identification of these ectoparasites.

A jiboia (Boa constrictor), vertebrado carnívoro, tem sido encontrada em abundância na área urbana de Manaus. A identificação morfológica dos carrapatos coletados em 27 dessas serpentes verificou a ocorrência de Amblyomma dissimile Koch 1844, em todos os exemplares avaliados e a presença de Amblyomma rotundatum Koch 1844, em duas dessas serpentes. A análise do marcador 16S rRNA confirma a identificação morfológica das espécies A. rotundatum e A. dissimile e apresenta novas sequências destes organismos.

Endoplasmic reticulum stress mediates lipopolysaccharide-induced apoptosis in rat hepatocyte / 法医学杂志

OBJECTIVE@#To investigate the role of endoplasmic reticulum stress (ERS) in lipopolysaccharide (LPS)-induced hepatocyte apoptosis.@*METHODS@#Cells of the rat hepatocyte line BRL were cultured. The hepatocytes were treated with LPS, ERS inducer thapsigargin (TG), and ERS inhibitor 4-phenylbutyric acid (4-PBA), respectively or in their different combination. The cell viability was measured by MTT assay. The cyto-nuclear morphological changes of apoptosis cells were detected by the fluorescent dye Hoechst 33258. The apoptosis rate was assessed by flow cytometry with Annexin V-FITC/PI double-staining. Expressions of GRP78 as ERS marker protein, CHOP, caspase-12 and cleaved-caspase-3 as ERS related protein were detected by Western blotting.@*RESULTS@#LPS could cause a decrease in cell viability and an increase in apoptosis rate in a dose- and time-dependent manner. The expression of GRP78, CHOP, caspase-12 and cleaved-caspase-3 proteins were significantly increased with LPS treatment. TG led to a marked decrease in cell viability and an increase in apoptosis rate, which aggravated the hepatocyte injury induced by LPS; whereas 4-PBA alleviated LPS-induced apoptosis.@*CONCLUSION@#ERS mediates LPS-induced hepatocyte injuries, indicating that ERS may play a vital role in the pathogenesis of LPS-induced hepatocyte injuries.

Inhibitory effect of ONO-AE3-208 on the formation of bone metastasis of prostate cancer in mice / 中华男科学杂志

<p><b>OBJECTIVE</b>To examine the effect of ONO-AE3-208, an EP4 antagonist, on the formation of bone metastasis from prostate cancer in mice.</p><p><b>METHODS</b>Thirty-four 6-week old nude mice were divided into an experimental and a control group of equal number to be treated by intraperitoneal injection of ONO-AE3-208 and double distilled water, respectively. Then PC3/LUC cells were constructed by stably transfecting luciferin to prostate cancer PC3 cells and inoculated into the left ventricle of the mice to establish an animal model of systemic bone metastasis. The time of metastasis formation, photon tumor burdens, and changes of the survival curves after modeling were compared between the two groups of mice.</p><p><b>RESULTS</b>At 30 days after modeling, bioluminescence imaging analysis showed that the photon tumor burdens were significantly increased in a time-dependent manner in the control group in comparison with those in the experimental group (P < 0.01). The rate of metastasis formation was significantly higher in the former than in the latter (93.3% vs 33.3%, P < 0.001). The median time of metastasis formation was 29 d (95% CI 26.547 - 35.262) in the experimental animals as compared with 21 d (95% CI 17.213 -24.787) in the controls (P < 0.001).</p><p><b>CONCLUSION</b>EP4 antagonist ONO-AE3-208 can inhibit the formation of bone metastasis from prostate cancer in mice.</p>

4-phenylbutyric Acid Regulates Collagen Synthesis and Secretion Induced by High Concentrations of Glucose in Human Gingival Fibroblasts

High glucose leads to physio/pathological alterations in diabetes patients. We investigated collagen production in human gingival cells that were cultured in high concentrations of glucose. Collagen synthesis and secretion were increased when the cells were exposed to high concentrations of glucose. We examined endoplasmic reticulum (ER) stress response because glucose metabolism is related to ER functional status. An ER stress response including the expression of glucose regulated protein 78 (GRP78), C/EBP homologous protein (CHOP), inositol requiring enzyme alpha (IRE-1alpha) and phosphoreukaryotic initiation factor alpha (p-eIF-2alpha) was activated in the presence of high glucose. Activating transcription factor 4 (ATF-4), a downstream protein of p-eIF-2alpha as well as a transcription factor for collagen, was also phosphorylated and translocalized into the nucleus. The chemical chaperone 4-PBA inhibited the ER stress response and ATF-4 phosphorylation as well as nuclear translocation. Our results suggest that high concentrations of glucose-induced collagen are linked to ER stress and the associated phosphorylation and nuclear translocation of ATF-4.

Effect of sodium phenylbutyrate on the apoptosis of human tongue squamous cancer cell line and expression of p21 and survivin genes / 中华口腔医学杂志

<p><b>OBJECTIVE</b>To examine the effects of sodium phenylbutyrate on the apoptosis of human tongue squamous cancer cell line and expression of p21 and survivin genes.</p><p><b>METHODS</b>The inhibition effects of sodium phenylbutyrate on Tca8113 and human tongue squamous cell carcinoma (TCSSA) cell lines were detected by methyl thiazoly terazolium (MTT) and the apoptosis of the cancer cells after being induced by sodium phenylbutyrate examined by flow cytometry (FCM). The expression of p21 and survivin genes were observed with Western blotting and RT-PCR.</p><p><b>RESULTS</b>Compared with control group, the level of p21 mRNA and protein of Tca8113 cellline increased to 0.09 ± 0.08 and increased 0.72 ± 0.10, that of TCSSA cellline increased 1.34 ± 0.12 and 1.56 ± 0.09 (P < 0.05). Compared with control group, the level of surrive mRNA and protein of Tca8113 cellline decreased to 1.10 ± 0.05 and 1.14 ± 1.10, that of TCSSA cellline decreased to 0.12 ± 0.08 and 0.94 ± 0.09 (P < 0.05). Sodium phenylbutyrate inhibited the cell proliferation, promoted cell apoptosis and arrested the cells in G₁/G₀ phase. The amount of p21 mRNA and protein were increased, and the expression of survivin gene was decreased.</p><p><b>CONCLUSIONS</b>Sodium phenylbutyrate exhibited remarkable inhibitory effects on human tongue squamous cancer cell proliferation and induced cancer cell apoptosis. The mechanism may be due to up-regulation of p21 gene and down-regulation of survivin gene. The mRNA level of p21 gene and survivin gene showed a strong correlation.</p>

Growth inhibition and gene induction in human hepatocellular carcinoma cell exposed to sodium 4-phenylbutanoate / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Sodium 4-phenylbutanoate (NaPB) can induce cellular differentiation and cell cycle arrest. However, its potential anticancer properties in hepatocellular carcinoma and influence on normal liver cell are still unclear. We observed the effects of NaPB on growth inhibition, including differentiation and phase growth arrest in normal liver cell line L-02 and hepatocellular carcinoma cell line Bel-7402. Furthermore, we investigated its mechanism in Bel-7402. METHODS; Hepatocellular carcinoma cells Bel-7402 and normal liver cell line L-02 were treated with NaPB at different concentrations. Light microscopy was used to find morphological change in cells. Cell cycle was detected by flow cytometry. Expression of acetylating histone H4 and of histones deacetylase 4 (HDAC4) were determined by Western blot. The expression of P21WAF1/CIP1 and E-cadherin were observed through immunocytochemistry.</p><p><b>RESULTS</b>NaPB treatment led to time dependent growth inhibition in hepatocellular carcinoma cells Bel-7402. NaPB treatment caused a significant decline in the fraction of S phase cells and a significant increase in G0/G1 cells. NaPB increased the expression of P21(WAF1/CIP1) and E-cadherin in Bel-7402 and significantly decreased the level of HDAC4 in Bel-7402. NaPB significantly improved the level of acetylating histone H4. The normal liver cell line L-02 showed no distinct changes under treatment with NaPB.</p><p><b>CONCLUSIONS</b>NaPB inhibited the growth of hepatocellular carcinoma cells Bel-7402 and induced partial differentiation through enhancing the acetylating histones. In Bel-7402, the expressions of P21(WAF1/CIP1) and E-cadherin may be related to level of acetylating histones and inhibition of cellular growth. NaPB showed no significant effect on normal liver cells.</p>

Phenol butyrate inhibits cell cycle of leukemia cell lines through up-regulation of p21WAF1/CIP1 gene / 中华血液学杂志

<p><b>OBJECTIVE</b>To elucidate effects of histone deacetylase inhibitors on cell cycle of leukemia cell lines and investigate its molecular mechanisms.</p><p><b>METHODS</b>Kasumi-1, U937 and NB4 cell lines were exposed to a histone deacetylase inhibitor, phenyl butyrate (PB), for 24, 48 and 72 hrs. Cells were harvested for cell cycle analysis by flow cytometry. Gene expression of p21WAF1/CIP1, a cyclin-dependent kinase inhibitor, was determined by semi-quantitative reverse transcriptase polymerase chain reaction (semi-quantitative RT-PCR). Promoter activity of p21WAF1/CIP1 was determined by luciferase-reporter assay in 293T cell line.</p><p><b>RESULTS</b>PB inhibited cell cycle of Kasumi-1, U937 and NB4 cell lines, showing G(0)/G(1) phase arrest and S-phase fraction reduction with a dose and time dependent manner. After Kasumi-1, U937 or NB4 cells exposed to 3 mmol/L PB for 72 hrs, G(0)/G(1)-phase fraction was increased by 42.03%, 44.36% and 26.82%, and S-phase fraction was decreased by 31.86%, 38.9% and 26.77%, respectively. After Kasumi-1, U937 and NB4 cell lines exposed to PB, the expression of p21WAF1/CIP1 gene was increased by (2.06 +/- 0.27), (2.78 +/- 0.40) and (1.78 +/- 0.20) times at its maximum, respectively. PB could stimulate p21WAF1/CIP1 promoter activity (by luciferase-reporter assay) and the effect was dose dependent. The promoter activity was increased by 5.74 times after the cells exposed to 3 mmol/L PB for 48 hrs. PB stimulating p21WAF1/CIP1 promoter activity was mainly mediated by a 101 base pairs fragment upstream of transcription start site.</p><p><b>CONCLUSION</b>PB could inhibit cell cycle of leukemia cell lines. The effects were mainly through up-regulation of p21WAF1/CIP1 expression.</p>

5-Aza-2'-deoxycytidine enhances differentiation and apoptosis induced by phenylbutyrate in Kasumi-1 cells / 中华肿瘤杂志

<p><b>OBJECTIVE</b>To investigate whether phenylbutyrate (PB) combined with 5-aza-2'-deoxycytidine (5-Aza-CdR)could inhibit transcription repression and induce t(8;21) acute myelogenous leukemia (AML) Kasumi-1 cells to differentiate and undergo apoptosis.</p><p><b>METHODS</b>Kasumi-1 cells were treated with PB and 5-Aza-CdR at different concentrations in suspension culture. Cellular proliferation was determined by the MTT assay, expression of myeloid-specific differentiation antigen and cell cycles were analyzed by flow cytometry. Cell apoptosis were assessed using AnnexinV/PI staining and flow cytometry.</p><p><b>RESULTS</b>Treatment of Kasumi-1 cells with PB caused a dose-dependent inhibition of proliferation, with an IC(50) of 2.3 mmol/L. When combined with 5-Aza-CdR, PB resulted in a greater growth inhibition with an IC(50) of 1.95 mmol/L. Treatment of Kasumi-1 cells with PB resulted in cell cycle arrest at G(0)/G(1), while combined treatment with PB and 5-Aza-CdR led to cell cycle arrest at G(2)/M. Expression of myeloid cell differentiation antigens CD11b and CD13 induced by PB was enhanced when Kasumi-1 cells were pretreated with low dose of 5-Aza-CdR. High, but not low, concentrations of 5-Aza-CdR could enhance early apoptosis of Kasumi-1 cells induced by PB.</p><p><b>CONCLUSION</b>Phenylbuty rate, when combined with 5-Aza-CdR, inhibits AML cell in vitro proliferation and increases apoptosis in a synergistic fashion.</p>

Combination of phenylbutyrate and 5-Aza-2'deoxycytidine inhibits human Kasumi-1 xenograft tumor growth in nude mice / 中华血液学杂志

<p><b>OBJECTIVE</b>To investigate the tumor suppression efficacy of histone deacetylase inhibitor, phenylbutyrate (PB), in combination with DNA methylation inhibitor 5-Aza-2-deoxycytidine (5-Aza-CdR) in the treatment of Kasumi-1 xenograft tumor in nude mice and its mechanism.</p><p><b>METHODS</b>The nude mice model of Kasumi-1 xenograft tumor was established by subcutaneous inoculation. Latency of tumor formation, the ability of Kasumi-1 cells pre treated with PB to form the xenograft tumor, and the tumor suppression activity of PB and 5-Aza-CdR by intraperitoneal injection in xenografted mice model were detected. Cell differentiation and cell cycle parameters of the tumor cells were analyzed by flow cytometry analysis, apoptosis by TUNEL in situ hybridization, and tumor microvessel density (MVD) by immunohistochemistry study.</p><p><b>RESULTS</b>The latency of tumor formation in mice with or without previous lienectomy was 17 approximately 23 and 40 approximately 50 days, respectively. Tumor cells xenografted could not be found in other tissues than in inoculation area, and still harbored the specific t(8;21) and AML1-ETO fusion gene. When the xenografted mice models treated with PB, 5-Aza-CdR, or both, the tumor growth inhibition rates were 49.07%, 25.69% and 87.46% (P < 0.05), the apoptosis indexes (AI) of tumor cells were (2.25 +/- 0.85)%, (1.32 +/- 0.68)%, and (5.41 +/- 1.56)% (P < 0.05), and the microvessel densities (MVD) were 21.69 +/- 6.25, 28.34 +/- 4.24 and 9.48 +/- 3.21 (P < 0.01), respectively. All the data above were significantly different from that in control (P < 0.05). The expression of CD11b and CD13 antigen of the tumor cells was increased in xenografted mice model treated with PB when compared with the control \[(12.08 +/- 1.02)% and (54.91 +/- 2.72)%\], respectively (P < 0.01), and tumor cells showed a cell cycle arrest with increased G(0)/G(1)-phase cells and decreased S-phase cells.</p><p><b>CONCLUSION</b>PB inhibited the growth of Kasumi-1 xenograft tumor by inducing tumor cell apoptosis and differentiation, and suppressing its angiogenesis in vivo. 5-Aza-CdR could significantly enhance the antitumor activity of PB.</p>

Effect of phenylbutyrate, a histone deacetylase inhibitor, on differentiation and apoptosis of Kasumi-1 cells / 中华血液学杂志

<p><b>OBJECTIVE</b>To explore the blockade effect of phenylbutyrate (PB), a histone deacetylase inhibitor, on the in vitro biological function of AML1/ETO to reverse its transcription repression and induce Kasumi-1 cells to differentiate and apoptosis.</p><p><b>METHODS</b>Kasumi-1 cells were treated with PB at different concentrations in suspension culture. Cell proliferation was analysed by MTT assay, morphological changes by light and electron microscopy, expression of myeloid-specific differentiation antigen and cell cycle by flow cytometry, cell apoptosis by annexin V staining, agarose gel electrophoresis and flow cytometry.</p><p><b>RESULTS</b>PB treatment caused a dose-dependent inhibition of the cell proliferation. The IC(50) was about 2.3 mmol/L. PB treatment led to a progressive decline in the fraction of S-phase cells and increase in G(0)/G(1) cells. PB induced a time- and dose-dependent increase in expression of myeloid cell surface protein CD(11b) and CD(13). A dose-dependent increase in early apoptosis for 2 days treatment, late apoptosis for 3 days treatment. The DNA ladder of apoptosis was observed on agarose gel electrophoresis for 5 days treatment. Morphological features of monocytoid differentiation and apoptosis were seen on Wright-Giemsa staining smears.</p><p><b>CONCLUSION</b>PB treatment could inhibit proliferation of Kasumi-1 cells, induce partial differentiation, apoptosis and accumulation of cells in G(0)/G(1) phase.</p>