Antitumor efficacy of FK228, a novel histone deacetylase inhibitor,depends on the effect on expression of angiogenesis factors

It has been recently demonstrated that histone deacetylase inhibitors inhibit angiogenesis, but their mechanism of action has not been characterized well. In this study, we examined the in vitro and in vivo effects of FK228 [(E)-(1S,4S,10S,21R)-7-[(Z)-ethylidene]-4,21-diisopropyl-2-oxa-12,13-dithia-5,8,20,23-tetraazabicyclo-[8,7,6]-tricos-16-ene-3,6,9,19,22-pentanone; FR901228, depsipeptide], an HDAC inhibitor, on the expression of angiogenesis factors in FK228-sensitive PC-3 prostate and FK228-resistant ACHN renal cancer cells. FK228 suppressed the expression of VEGF mRNA in PC-3 cells, but not in ACHN cells. FK228 also suppressed the expression of basic fibroblast growth factor (bFGF) mRNA in both PC-3 and ACHN cells. Under conditions of hypoxia, FK228 suppressed the expression of VEGF mRNA without modulating the expression of hypoxia-inducible factor-1 alpha mRNA in PC-3 cells. FK228 induced the highest acetylation of histone H3 and H4 in the P2 region of the VEGF promoter, which includes the hypoxia-inducible factor-1 alpha binding site that plays an important role in regulating the expression of VEGF gene. Moreover, FK228 reduced the amount of VEGF and bFGF protein, and their mRNA levels in PC-3 xenograft implanted in nude mice, but did not reduce them in ACHN xenograft. In conclusion: (i) FK228 showed a suppressive effect on the expression of angiogenesis factors, such as VEGF and bFGF, in PC-3 xenograft but not in ACHN xenograft, which suggests that the effect on the expression of angiogenesis factors is important for the antitumor efficacy of FK228; (ii) FK228 caused histone acetylation of the VEGF promoter regions, which may contribute to the suppression of VEGF gene expression.     

Supernatant of bacterial fermented soybean induces apoptosis of human hepatocellular carcinoma Hep 3B cells via activation of caspase 8 and mitochondria.

SC-1, the aqueous phase of soybean fermentation products by bacteria (Bacillus subtilis and Bacillus brevis), significantly inhibited the growth and clonogenesity of human hepatocellular (Hep 3B), mouse hepatocellular (ML-1), and human colorectal (HCT 116 and HT-29) carcinoma cells. Cytotoxicity of SC-1 in Hep 3B cells was through the process of apoptosis characterizing by increase in cell population of sub-G(1) phase, fragmentation of DNA, and change of nuclear morphology. Treatment of Hep 3B cells with SC-1 activated caspase 8 and caspase 3. Elevation of nuclear DNA fragmentation factor 40 (DFF40) and cleavage form of poly(ADP-ribose) polymerase (PARP) were also observed. SC-1 also activated intrinsic pathway via increase of pro-apoptotic (tBid, Bak and Bax) and decrease of anti-apoptotic (Bcl-2 and Bcl-x(L)) proteins on mitochondria, disruption of mitochondrial membrane potential, release of cytochrome c and Smac (second mitochondria-derived activator of caspase/direct IAP binding protein with low PI) from mitochondria, and activation of caspase 9. Inhibition on protein expression of Ku70 in cytosol and cyclooxygenase (COX)-2, but not COX-1, in whole cell lystes were revealed in SC-1-treated Hep 3B cells. These results suggest caspase 8, Ku70 and mitochondria are involved in the antitumor mechanism of SC-1 in Hep 3B cells.     

Suppression of mitogen-activated protein kinase phosphatase-1 (MKP-1) by heparin in vascular smooth muscle cells

Heparin inhibits vascular smooth muscle cell (VSMC) proliferation, but mechanisms remain elusive. Because heparin inhibits signaling through multiple kinase cascades, we investigated the possibility that phosphatases could be involved. Mitogen-activated protein kinase phosphatase-1 (MKP-1) was the predominant MKP detected in VSMC lines. MKP-1 protein was increased by serum stimulation of quiescent cells, and this increase was diminished by heparin (1 microg/mL). Increased MKP-1 expression was dependent on the mitogen-activated protein kinase, Erk. Decreased Erk activity in the presence of heparin preceded, and may account for, decreased MKP-1. The antimitogenic effects of heparin are therefore unlikely to act through a shift in the kinase/phosphatase balance, but rather through direct kinase suppression. However, because MKP-1 is known to cause an increase in activity of kinases upstream of Erk, that may signal through additional pathways, the decrease in MKP-1 activity may paradoxically enhance heparin's antiproliferative effects. VSMC selected to grow in the presence of heparin express decreased levels of MKP-1 that are unresponsive to heparin, and Erk activity becomes unresponsive to heparin in one cell line. We conclude that phosphatase activation is not a direct mechanism of suppression of multiple kinase cascades by heparin.     

羟基红花黄色素A对氧化低密度脂蛋白诱导的血管平滑肌细胞增殖的抑制作用

目的探讨羟基红花黄色素A(HSYA)抑制氧化低密度脂蛋白(ox-LDL)诱导血管平滑肌细胞(VSMC)增殖与细胞外信号调节激酶1/2(ERK1/2)和丝裂原活化蛋白激酶磷酯酶-1(MKP-1)基因转录及蛋白表达之间的关系。方法 HSYA 10μmol.L-1和ox-LDL 35 mg.L-1与VSMC共培育48 h,MTT检测细胞存活率,流式细胞术检测细胞周期,逆转录PCR(RT-PCR)观察ERK1/2和MKP-1基因表达,Western印迹法观察ERK1/2和MKP-1蛋白表达。结果与ox-LDL 35 mg.L-1模型组比较,加入HSYA 10μmol.L-1组细胞存活率明显下降,G0/G1期细胞明显增多,ERK1 mRNA表达下降了42.2%(P<0.01),MKP-1 mRNA显著增加了86.9%(P<0.01),p-ERK1/2蛋白表达降低了39.9%(P<0.01),MKP-1蛋白表达增加了80.6%(P<0.01);加入PD98059 10μmol.L-1组,细胞存活率明显降低了58.3%(P<0.01),G0/G1期细胞明显增多(P<0.01),ERK1 mRNA表达下降了45.9%(P<0.01);p-ERK1/2蛋白表达下降了45.9%(P<0.01);PD98059 10μmol.L-1+HSYA 10μmol.L-1同时加入,细胞生存率下降了61.9%(P<0.01),G0/G1期细胞明显增多(P<0.01),ERK1 mRNA表达进一步减少(P<0.01),MKP-1 mRNA增加了110%(P<0.01),p-ERK1/2蛋白表达下降了51.2%(P<0.01),MKP-1蛋白表达增加了62.4%(P<0.01)。结论 HSYA通过增强MKP-1蛋白表达、降低p-ERK1/2蛋白活性和抑制细胞周期运转的机制抑制VSMC增殖。     

Effects of FK228, a novel histone deacetylase inhibitor,on human lymphoma U-937 cells in vitro and in vivo

FK228 [(E)-(1S,4S,10S,21R)-7-[(Z)-ethylidene]-4,21-diisopropyl-2-oxa-12,13-dithia-5,8,20,23-tetraazabicyclo-[8,7,6]-tricos-16-ene-3,6,9,19,22-pentanone; FR901228, depsipeptide] is a novel histone deacetylase inhibitor that shows therapeutic efficacy in Phase I trials of patients with malignant lymphoma. However, its mechanism of action has not been characterized. In this study, we examined the in vitro and in vivo effects of FK228 on human lymphoma U-937 cells. FK228 very strongly inhibited the growth of U-937 cells with an IC(50) value of 5.92 nM. In a scid mouse lymphoma model, mice treated with FK228 once or twice a week survived longer than control mice, with median survival times of 30.5 (0.56 mg/kg) and 33 days (0.32 mg/kg), respectively (vs. 20 days in control mice). Remarkably, 2 out of 12 mice treated with FK228 (0.56 mg/kg once or twice a week) survived past the observation period of 60 days. The apoptotic population of U-937 cells time-dependently increased to 37.7% after 48 hr of treatment with FK228. In addition, FK228 induced G1 and G2/M arrest and the differentiation of U-937 cells to the CD11b(+)/CD14(+) phenotype. Expression of p21(WAF1/Cip1) and gelsolin mRNA increased up to 654- and 152-fold, respectively, after 24hr of treatment with FK228. FK228 caused histone acetylation in p21(WAF1/Cip1) promoter regions, including the Sp1-binding sites. In conclusion, (i) FK228 prolonged the survival time of scid mice in a lymphoma model, and (ii) the beneficial effects of FK228 on human lymphoma may be exerted through the induction of apoptosis, cell cycle arrest, and differentiation via the modulation of gene expression by histone acetylation.     

Diclofenac induces apoptosis in hepatocytes by alteration of mitochondrial function and generation of ROS

Diclofenac is a non-steroidal anti-inflammatory drug that is widely used clinically but side effects associated with the administration of the drug have been reported. The apoptotic effect of the drug has been evaluated in human and rat hepatocytes. Apoptosis was observed after exposure to sub-cytotoxic concentrations of the drug, without overlapping with cell necrosis. Flow cytometric analysis revealed a time- and dose-dependent increase of apoptotic nuclei with sub-diploid DNA content. Caspase 8 and 9 mediate the cell-receptor and the mitochondria-initiated apoptotic pathways, respectively. Inhibition of both caspases prevented activation of downstream caspases, thus indicating that diclofenac at least activates caspase 3 and both effector caspases 8 and 9. The hierarchy of caspase activation by diclofenac was investigated. Analysis of kinetics revealed a simultaneous activation of these caspases that was maximal after 12 hr of exposure to the drug. Inhibitors of MPT, prevented the downstream activation of the caspase cascade, thus showing that diclofenac opened the mitochondrial pore. On the other hand, antioxidants were able to prevent caspase activation by diclofenac, revealing that oxidative stress at the mitochondrial level is in the root of MPT induction and caspase cascade activation. Caspase activation is not mediated by Bid cleavage, suggesting that the cell-receptor pathway seems not to be involved. However, a dose-dependent release of caspase 8 from the mitochondria was observed, indicating that caspase 8 can be processed independently of cell death receptors. Caspases 8 and 9 are very likely the apical caspases in diclofenac-induced apoptosis. In addition, an early dose-dependent increase of bclX(L) expression parallel to the generation of reactive oxygen species in the mitochondria was found. In conclusion, the mitochondrial pathway is very likely the only pathway involved in diclofenac-induced apoptosis, which was related to CYP-mediated metabolism of diclofenac, with the highest apoptotic effect produced by the metabolite 5OH-diclofenac.     

地塞米松诱导乳腺癌细胞对紫杉醇耐药的研究

目的:探讨紫杉醇(PTX)对不同浓度地塞米松(DEX)预处理人乳腺癌细胞MCF-7耐药及丝裂原活化蛋白激酶磷酸酯酶-1(MKP-1)mRNA表达的影响。方法:四唑盐(MTT)试验测定紫杉醇对MCF-7细胞的存活率,筛选出药物的半数抑制浓度(IC50)。用不同浓度地塞米松预处理MCF-7细胞,再予紫杉醇作用于MCF-7细胞,MTT试验测定细胞存活率,RT-PCR法检测细胞中MKP-1 mRNA的表达水平。结果:不同浓度的地塞米松预处理MCF-7细胞后能诱导其对紫杉醇产生耐药,细胞耐药率及MKP-1 mRNA的表达水平随着地塞米松浓度的增加而增高。结论:地塞米松预处理MCF-7细胞后能诱导其对紫杉醇产生耐药,其机制可能与MKP-1抑制细胞凋亡而介导乳腺癌耐药有关。     

Identification of gene expression profiles predicting tumor cell response to L-alanosine

The methylthioadenosine phosphorylase (MTAP) gene gained considerable interest as therapeutic target for tumors with the 9p21 deletion. This gene maps to 9p21 and loss of this chromosomal region in tumors offers an unique opportunity for chemoselective treatment, since MTAP is an important salvage enzyme for the formation of adenine that is needed for DNA synthesis. L-Alanosine, an antibiotic from Streptomyces alanosinicus, blocks the common de novo purine biosynthesis pathway and, thereby, inhibits tumor cells with MTAP deficiency. Normal cells escape the detrimental effects of L-alanosine due to their proficiency in the MTAP salvage pathway. The present analysis was undertaken to gain insights into the molecular architecture of tumor cells that determines the response to L-alanosine apart from the MTAP gene. Analysis of cell doubling times and IC(50) values for L-alanosine showed that slowly growing cell lines were more resistant to L-alanosine than rapidly growing ones. Mining the database of the National Cancer Institute (N.C.I.), for the mRNA expression of 9706 genes in 60 cell lines by means of Kendall's tau-test, false discovery rate calculation, and hierarchical cluster analysis pointed to 11 genes or expressed sequence tags whose mRNA expression correlated with the IC(50) values for L-alanosine. Furthermore, we tested L-alanosine for cross-resistance in multidrug-resistant cell lines which overexpress selectively either the P-glycoprotein/MDR1 (CEM/ADR5000), MRP1 (HL-60/AR), or BCRP (MDA-MB-231-BCRP) genes. None of the multidrug-resistant cell lines was cross-resistant to L-alanosine indicating that L-alanosine may be suitable to treat multidrug-resistant, refractory tumors in the clinic. Finally, the IC(50) values for L-alanosine of the 60 cell lines were correlated to the p53 mutational status and expression of p53 downstream genes. We found that p53 mutated cell lines were more resistant to L-alanosine than p53 wild type cell lines.     

Resistance to Fas-mediated apoptosis in malignant tumours is rescued by KN-93 and cisplatin via downregulation of c-FLIP expression and phosphorylation

1. The purpose of the present study was to investigate the molecular mechanisms that control tumour cell resistance and to search for molecules that could overcome Fas ligand (FasL) or CH-11 resistance in certain tumours, including glioma and melanoma. 2. Twelve tumour cell lines were examined for their sensitivity to CH-11-induced apoptosis and then two of each of the CH-11-sensitive and -resistant tumour cell lines were analysed for Fas-mediated death-inducing signalling complex (DISC). The calmodulin kinase II (CaMKII) inhibitor KN-93 and the chemotherapeutic drug cisplatin were used to treat resistant cells; the effects of these two drugs on CH-11-resistant tumour cells were investigated. 3. In CH-11-sensitive tumour cells, apoptosis-initiating caspase 8 and caspase 10 were recruited to the DISC, where they became activated through autocatalytic cleavage, leading to apoptosis through cleavage of downstream substrates, such as caspase 3 and DNA fragmentation factor 45. 4. In CH-11-resistant cells, cellular Fas-associated death domain-like interleukin-1b-converting enzyme inhibitory protein (c-FLIP) proteins were recruited to the DISC, resulting in inhibition of caspase 8 and caspase 10 cleavage. The c-protein expression and phosphorylation of FLIP and CaMKII protein and enzyme activity were upregulated in resistant cells. Treatment of resistant cells with 100 micromol/L KN-93 and 10 microg/mL cisplatin downregulated c-FLIP expression, inhibited c-FLIP phosphorylation and rescued CH-11 sensitivity. 5. In conclusion, KN-93 and cisplatin inhibit c-FLIP protein expression and phosphorylation restores CH-11-induced apoptosis in tumour cells. tHe present study provides evidence for the use of a new combination therapeutic strategy in the treatment of malignant tumours.     

Effects of sodium ferulate on amyloid-beta-induced MKK3/MKK6-p38 MAPK-Hsp27 signal pathway and apoptosis in rat hippocampus

AIM: To observe the effects of sodium ferulate (SF) on amyloid beta (Abeta)1-40-induced p38 mitogen-activated protein kinase (MAPK) signal transduction pathway and the neuroprotective effects of SF. METHODS: Rats were injected intracerebroventricularly with Abeta1-40. Six hours after injection, Western blotting was used to determine the expressions of phosphorylated mitogen-activated protein kinase kinase (MKK) 3/MKK6, phospho-p38 MAPK, interleukin (IL)-1beta, phospho-MAPK activating protein kinase 2 (MAPKAPK-2), the 27 kDa heat shock protein (Hsp27), procaspase-9, -3, and -7 cleavage, and poly (ADP-ribose) polymerase (PARP) cleavage. Seven days after injection, Nissl staining was used to observe the morphological change in hippocampal CA1 regions. RESULTS: Intracerebroventricular injection of Abeta1-40 induced an increase in phosphorylated MKK3/MKK6 and p38 MAPK expressions in hippocampal tissue. These increases, in combination with enhanced interleukin (IL)-1beta protein expression and reduced phospho-MAPKAPK2 and phospho-Hsp27 expression, mediate the Abeta-induced activation of cell death events as assessed by cleavage of procaspase-9, -3, and -7 and caspase-3 substrate PARP cleavage. Pretreatment with SF (100 mg/kg and 200 mg/kg daily, 3 weeks) significantly prevented Abeta1-40-induced increases in phosphorylated MKK3/MKK6 and p38 MAPK expression. The Abeta1-40-induced increase in IL-1beta protein level was attenuated by pretreatment with SF. In addition, Abeta1-40-induced decreases in phosphorylated MAPKAPK2 and Hsp27 expression were abrogated by administration of SF. In parallel with these findings, Abeta1-40-induced changes in activation of caspase-9, caspase-7, and caspase-3 were inhibited by pretreatment with SF. CONCLUSION: SF prevents Abeta1-40-induced neurotoxicity through suppression of MKK3/MKK6-p38 MAPK activity and IL-1beta expression and upregulation of phospho-Hsp27 expression.     

Reduction of metastasis, cell invasion, and adhesion in mouse osteosarcoma by YM529/ONO-5920-induced blockade of the Ras/MEK/ERK and Ras/PI3K/Akt pathway

Osteosarcoma is one of the most common primary malignant bone tumors in children and adolescents. Some patients continue to have a poor prognosis, because of the metastatic disease. YM529/ONO-5920 is a nitrogen-containing bisphosphonate that has been used for the treatment of osteoporosis. YM529/ONO-5920 has recently been reported to induce apoptosis in various tumors including osteosarcoma. However, the mode of metastasis suppression in osteosarcoma by YM529/ONO-5920 is unclear. In the present study, we investigated whether YM529/ONO-5920 inhibited tumor cell migration, invasion, adhesion, or metastasis in the LM8 mouse osteosarcoma cell line. We found that YM529/ONO-5920 significantly inhibited metastasis, cell migration, invasion, and adhesion at concentrations that did not have antiproliferative effects on LM8 cells. YM529/ONO-5920 also inhibited the mRNA expression and protein activities of matrix metalloproteinases (MMPs). In addition, YM529/ONO-5920 suppressed phosphorylated extracellular signal-regulated kinase 1/2 (ERK1/2) and the serine/threonine protein kinase B (Akt) by the inhibition of Ras prenylation. Moreover, U0126, a mitogen-activated protein kinase kinase (MEK) 1/2 inhibitor, and LY294002, a phosphatidylinositol 3-kinase (PI3K) inhibitor, also inhibited LM8 cell migration, invasion, adhesion, and metastasis, as well as the mRNA expression and protein activities of MMP-1, MMP-2, MMP-9, and MT1-MMP. The results indicated that YM529/ONO-5920 suppressed the Ras/MEK/ERK and Ras/PI3K/Akt pathways, thereby inhibiting LM8 cell migration, invasion, adhesion, and metastasis. These findings suggest that YM529/ONO-5920 has potential clinical applications for the treatment of tumor cell metastasis in osteosarcoma.     

Coffea arabica extract and its constituents prevent photoaging by suppressing MMPs expression and MAP kinase pathway

UV is a potent factor in skin photoaging and photocarcinogenesis. Therefore, investigating the inhibiting mechanisms of photoaging would be useful to enable development of agents to slow down the aging process. UV-irradiation increased metalloproteinase (MMP)-1, -3, and -9 and then causes collagen and elastin degradation, leading to the formation of coarse wrinkles and sagging skin. Polyphenols, a group of compounds, possessing a variety of biological activities including inhibition of MMP-1 and elastase, are widely distributed in plants including Coffea arabica. In this study, Coffea arabica leaves extract (CAE), its hydrolysates (CAH), chlororgenic acid and caffeic acid, are studied for their anti-photoaging effect. Coffea arabica leaves were extracted with methanol, and the extract was hydrolyzed with different concentrations of hydrochloric acid. The various concentrations of CAE, CAH, chlororgenic acid and caffeic acid were subject to MMPs and elastase inhibition tests. The fibroblast was used for collagen synthesis and MMP-1, -3, -9 inhibition tests on herbal extracts. The results showed that CAE stimulated type I procollagen expression, inhibited MMP-1, -3, -9 expression and inhibited the phosphorylation of JNK, ERK and p38. The results suggest that CAE can prevent photo-damage in skin through inhibiting MMP expression and MAP kinase pathway.     

Prevention of macrophage adhesion molecule-1 (Mac-1)-dependent neutrophil firm adhesion by taxifolin through impairment of protein kinase-dependent NADPH oxidase activation and antagonism of G protein-mediated calcium influx

Taxifolin has been reported to down-regulate the expression of intercellular adhesion molecule-1 (ICAM-1), a receptor-mediating firm adhesion with beta2 integrin (e.g., Mac-1) expressed on leukocytes. To evaluate whether taxifolin could modulate Mac-1-dependent firm adhesion by neutrophils, and the possible mechanism(s) underlying its anti-inflammatory action, its effects on N-formyl-methionyl-leucyl-phenylalanine (fMLP) or phorbol-12-myristate-13-acetate (PMA)-activated peripheral human neutrophils were studied. Pretreatment with taxifolin (1-100 microM) concentration-dependently diminished fMLP- or (PMA)-induced Mac-1-dependent firm adhesion and upexpression of surface Mac-1. Mobilisation of intracellular calcium and production of reactive oxygen species (ROS) signal the upexpression of Mac-1 and firm adhesion by neutrophils. Taxifolin impeded the calcium influx induced by fMLP (a receptor-mediated activator) or AlF(4)(-) (a G protein-mediated activator). Taxifolin also effectively inhibited the fMLP- or PMA-induced ROS production with 50% inhibitory concentration (IC(50)) less than 10microM, possibly through impairing the activation of NADPH oxidase, a major ROS-generating enzyme in neutrophils, by restricting the activation of p38 mitogen-activated protein kinase (p38 MAPK) and protein kinase C (PKC). In conclusion, we propose that impairment of ROS production by NADPH oxidase through interfering with p38 MAPK- and/or PKC-dependent signals, and antagonism of G protein-mediated calcium influx may account for the inhibition of Mac-1-dependent neutrophil firm adhesion that confers taxifolin the anti-inflammatory activity.     

DNA methylation changes of apoptotic genes in organogenesis stage of mice embryos by maternal chlorpyrifos induction

The continued use of pesticides is one of the requirements of modern agriculture. Investigations have shown that pesticides can alter gene methylation and expression and subsequently may lead to abortion or birth of embryos with teratogenic disorders. In present study, 30 female NMRI mouse were divided in three experimental groups which in the CPF group, intraperitoneal chlorpyrifos was injected, in the sham group, DMSO was injected, and the control group without injection. The mice were mated and utinized 10 days' post gestation. The number of embryos in each fertilized female, maternal weight, and liver fibrosis was evaluated. The apoptosis pathway genes (caspase3, caspase9) and protein expressions (pro‐caspase3, caspase3) of the embryos were evaluated with qRT‐PCR and western blot, respectively. The DNA methylation of caspase3 and caspase9 were also assessed. The number of embryos and obtained maternal weight from the CPF group was significantly lower than other two groups. The mRNA expression of Caspase3 and Caspase9 were significantly higher in the CPF group. The protein expression evaluation confirmed the results achieved at the mRNA level. The percentage of Caspase9 DNA methylation in embryos collected from the CPF group was higher compared to the others. It can be considered that consumption of chlorpyrifos toxin can alter the DNA methylation and increase the expression of apoptotic genes. Therefore, continuous use of chlopyrifos may affect pregnancy by increasing the apoptosis pathway in the developing embryos which may lead to abortion or teratogenic disorders in newborn infants.     

In rat renal fibroblasts, mycophenolic acid inhibits proliferation and production of the chemokine CCL2, stimulated by tumour necrosis factor-��

BACKGROUND AND PURPOSE

Renal fibroblasts play a pivotal role in the development of tubulointerstitial fibrosis, a condition highly predictive of progression towards end-stage renal disease. The present study investigated the anti-mitogenic and anti-inflammatory effects of an inhibitor of inosine monophosphate dehydrogenase, mycophenolic acid (MPA) and the mechanisms underlying its action in normal rat kidney fibroblasts (49F cells).

EXPERIMENTAL APPROACH

Proliferation of 49F cells was studied by tetrazole 3-(4, 5-dimethylthiazol-2-yl-)-2,5-diphenyltetrazolium bromide (MTT) test, bromodeoxyuridine incorporation and flow cytometry. The cyclins, tumour suppressor genes and phospho-mitogen-activated protein kinases (MAPKs) were semiquantified by immunoblotting. Apoptosis was measured by quantifying the fragmented DNA and the activity of caspase 3. The monocyte chemokine CCL2 was measured by ELISA. The mRNA expression of CCL2 was measured by real-time PCR.

KEY RESULTS

Mycophenolic acid dose-dependently inhibited steady-state proliferation of 49F cells by up-regulation of p21, p27 and p53, in association with a decrease in cyclins D2 and E. Treatment with MPA also triggered apoptosis of 49F cells by activating the caspase 3 cascade. Furthermore, MPA attenuated tumour necrosis factor-α-induced CCL2 expression through down-regulation of p38 MAPK, but not that of ERK1/2 or JNK.

CONCLUSIONS AND IMPLICATIONS

The anti-mitogenic and anti-inflammatory effects of MPA were mediated by up-regulation of cell cycle inhibitors and pro-apoptotic signals, and by suppression of p38 MAPK pathway respectively. This dual effect of MPA may form the rationale for animal or clinical trials for the treatment of fibrotic renal diseases.     

FLIP、Caspese 8及MTA1在上皮性卵巢肿瘤中的表达及其临床意义

目的：研究凋亡抑制蛋白FLIP，Caspase8，肿瘤转移相关基因MTA1在上皮性卵巢肿瘤中表达及其意义，探讨它们与卵巢癌临床病理指标之间的关系。方法：应用免疫组化SP法，检测FLIP、Caspase8、MTA1在98例上皮性卵巢肿瘤（包括18例良性囊腺瘤，20例变界性囊腺瘤和60例卵巢癌）的表达情况。结果：FLIP、Caspase8及MTA1在卵巢交界性囊腺瘤和卵巢癌中的表达均分别明显高于卵巢良性囊腺瘤（P〈0．05），FLIP、MTA1的表达在组织学分型、分化程度之间无显著性差异（P〉0．05）。而与卵巢癌的FIGO分期及有无转移之间有显著性差异（P〈0．05）。并且FLAP和Caspase8成负相关关系。结论：FLIP、MTA1在卵巢癌中的发生发展中起重要作用。期望能为判断卵巢癌的生物学行为，决定治疗方案提供理论依据。     

Adenosine modulates vascular endothelial growth factor expression via hypoxia-inducible factor-1 in human glioblastoma cells

Hypoxia appears to induce a program which shifts the cellular phenotype toward an increase in extracellular adenosine. Hypoxia-inducible factor-1 (HIF-1) is a key regulator of genes crucial to many aspects of cancer biology. Since in gliomas there is a strong correlation between HIF-1alpha expression, tumor grade and tumor vascularization, the aim of this study was to investigate whether adenosine may regulate HIF-1 in human glioblastoma cell lines. The results indicate that in the human hypoxic A172 and U87MG glioblastoma cell lines adenosine up-regulates HIF-1alpha protein expression via the A(3) receptor subtype. In particular, we investigated the effect of A(3) receptor antagonists on HIF-1 and vascular endothelial growth factor (VEGF) expression. We found that A(3) antagonists inhibit adenosine-induced HIF-1alpha and VEGF protein accumulation in the hypoxic cells. Investigations in the molecular mechanism showed that A(3) receptor stimulation activates p44/p42 and p38 MAPKs that are required for A(3)-induced increase of HIF-1alpha and VEGF. Further studies are required to demonstrate the in vivo relevance of these observations with regard to the proposed role for adenosine as a key element in hypoxia and in tumors.