Tramadol use is associated with enhanced postoperative outcomes in breast cancer patients: a retrospective clinical study with in vitro confirmation.

BACKGROUND: There is growing interest in the effect of postoperative analgesics on oncological outcomes after cancer surgery. We investigated the impact of tramadol after breast cancer surgery on recurrence and mortality and explored the mechanism by which tramadol affects cultured breast cancer cells in vitro. METHODS: Electronic medical records of patients who underwent breast cancer surgery between November 2005 and December 2010 at Severance Hospital in Korea were reviewed. Cox regression analyses were used to identify factors related to postoperative recurrence and mortality. We performed the sensitivity test with propensity score matching to adjust for selection bias. In addition, we investigated the effects of tramadol on human breast adenocarcinoma (Michigan Cancer Foundation-7 [MCF-7]) cells via assessment of cell viability, clonogenic assay, and cell cycle analysis in vitro. RESULTS: Of 2588 breast cancer patients, 36.4% had received tramadol. Those who received tramadol had a 0.71-fold decreased risk of recurrence and a 0.56-fold decrease in mortality. The MCF-7 cell viability assays showed that tramadol had an anti-proliferative effect by cell cycle arrest, suppressing colony formation, and regulation of oestrogen and progesterone receptors. Tramadol induced apoptosis of MCF-7 cells via extracellular signal-regulated kinases by decreasing of 5-hydroxytryptamine (HT)2B receptor and transient receptor potential vanilloid-1 expression. CONCLUSIONS: After breast cancer surgery, patients who received tramadol had a decreased risk of postoperative recurrence and mortality. The anti-tumour effect of tramadol appears to involve inhibition of proliferation, induction of apoptosis, and effects on 5-HT2B receptor and TRPV-1.

Effects of deep vs moderate neuromuscular block on the quality of recovery after robotic gastrectomy.

BACKGROUND: It remains unclear whether deep neuromuscular blockade results in better postoperative recovery than does moderate neuromuscular blockade. Therefore, in this study, we aimed to compare the effects of deep neuromuscular blockade and moderate neuromuscular blockade on the quality of postoperative recovery in patients undergoing robotic gastrectomy. METHODS: In this prospective, double-blind, single-center randomized controlled superiority trial with two parallel groups, 56 adult patients (19-80 years) scheduled for elective robotic gastrectomy were randomly assigned to a moderate neuromuscular blockade group or a deep neuromuscular blockade group in a 1:1 ratio. In the deep and moderate neuromuscular blockade groups, the infusion rate for rocuronium was adjusted to maintain a post-tetanic count of 1-2 or a train-of-four count of 1-2, respectively. The primary outcome was the Quality of Recovery-40 (QoR-40) score on postoperative day 1. Secondary outcomes included the QoR-40 score on postoperative day 2, intraoperative hemodynamic data, intraoperative respiratory data, visual analog scale score for pain, postoperative incidences of nausea and vomiting, postoperative rescue analgesic use, and postoperative rescue antiemetic use. RESULTS: The postoperative QoR-40 score was similar between the two groups on postoperative days 1 and 2. Moreover, the two groups showed no differences in intraoperative hemodynamic and respiratory data or postoperative pain, nausea and vomiting, and rescue medication use. CONCLUSION: Our findings suggest that the quality of recovery after robotic gastrectomy is similar for deep and moderate neuromuscular blockade. Therefore, deep neuromuscular blockade during robotic gastrectomy may be unnecessary, at least in patients with normal body mass index.

Comparison of the laryngeal mask airway supreme and the i-gel in paralysed elderly patients: A randomised controlled trial.

BACKGROUND: The laryngeal mask airway supreme (LMA-S) and i-gel are both popular second-generation supraglottic airway devices that have been widely studied in surgical patients, but their differences in clinical performance in the elderly are not clear. OBJECTIVE: We compared the efficacy and safety of the LMA-S and i-gel in anaesthetised and paralysed elderly patients. DESIGN: A randomised study. SETTING: Single-centre trial, study period January 2014 from to October 2016. PATIENTS: One hundred and six elderly patients who underwent urological or orthopaedic surgery with an expected duration less than 2 h. INTERVENTION: Patients were allocated to either the LMA-S (n = 53) or i-gel (n = 53) group. All insertions were performed in a standardised manner according to the manufacturers' instructions. MAIN OUTCOME MEASURES: Our primary endpoint was the rate of successful insertion at the first attempt. The adequacy of positive pressure ventilation and airway sealing, fibreoptic laryngoscopy grades and stability of airway maintenance during anaesthesia were also assessed. RESULTS: Although the rate of successful insertion at the first attempt was similar between the two groups (94.3 vs. 82.7%, P = 0.072), more patients required device manipulation during insertion with the LMA-S than the i-gel (42.3 vs. 18.9%, P = 0.011). Good fibreoptic laryngoscopy grades were significantly more common with the i-gel than the LMA-S (79.3 vs. 55.8%, P = 0.042), and peak inspiratory pressures were lower in the i-gel group both immediately after insertion and at the end of surgery. Leak pressures were significantly higher in the i-gel group than the LMA-S group, both immediately after insertion and at the end of surgery (25.8 vs. 23.0, P = 0.036; and 28.1 vs. 23.7, P < 0.001, respectively). CONCLUSION: Both the LMA-S and i-gel were used successfully and safely in elderly patients. However, the i-gel demonstrated better airway sealing than the LMA-S at insertion and during maintenance of anaesthesia. TRIAL REGISTRATION: NCT02026791 at clinicaltrial.gov.

Incidence of bowel injury during gynecologic surgery for benign indications: A nationwide cross-sectional study of cases from 2009 to 2018.

OBJECTIVE: To investigate the incidence and risk factors of intestinal injury during gynecologic surgery for benign diseases, based on a national database. METHOD: The study cohort was generated by extracting patients with operation codes for benign gynecologic diseases from the Health Insurance Review & Assessment Service National Inpatient Sample from 2009 to 2018. After analyzing the incidence of bowel injury during gynecologic surgery, a multivariate analysis was performed to identify the associated risk factors for bowel injury. RESULTS: Among 81 451 patients who underwent gynecologic surgery for benign diseases, the incidence of bowel injury was 6.14 per 1000 women. The risk of bowel injury decreased with laparoscopy (odds ratio [OR] 0.54; 95% confidence interval [CI] 0.41-0.69; P < 0.001) and increased with subtotal hysterectomy (OR 2.83; 95% CI 1.79-4.46; P < 0.001) and adnexectomy (OR 2.83; 95% CI 1.93-4.16; P < 0.001). Old age, higher Charlson comorbidity index, low socioeconomic status, and a higher clinic grade were associated with a higher risk of bowel injury. CONCLUSION: This study revealed the incidence of bowel injury during benign gynecologic surgery in a Korean national population-based cohort. The risk of bowel injury increased with open surgery, subtotal hysterectomy, and adnexectomy.

A HIF-1alpha-dependent autocrine feedback loop promotes survival of serum-deprived prostate cancer cells.

BACKGROUND: We previously reported that normoxic, serum-deprived prostate cancer (PCa) cells upregulate hypoxia-inducible factor-1alpha (HIF-1alpha) protein, which promotes survival during serum deprivation via insulin-like growth factor-2 (IGF-2) upregulation. This study investigated the molecular mechanism of autocrine regulation of HIF-1alpha, IGF-2 and cell survival in serum-deprived PC-3 and LNCaP PCa cells. METHODS: Cell viability was assessed by trypan blue assay. PI3K activity was inhibited with LY294002, and PTEN overexpression. mRNA was assessed by RT-PCR, and IGF-2 protein by ELISA. Activated insulin-like growth factor-I receptor (IGF-IR) was detected by probing immunoprecipitated IGF-IR for phospho-tyrosine. IGF-IR activity was inhibited with IGF-2 neutralizing antibody and IGF-IR-specific siRNA. HIF-1alpha, phospho-Akt, total-Akt and IGF-IR protein was assessed by immunoblots. HIF-1alpha was suppressed with siRNA. RESULTS: We detected a time-dependent increase in Akt activation during serum deprivation, and inhibition of Akt activation attenuated the serum deprivation-mediated increase in HIF-1alpha and cell survival. Importantly, IGF-2 secretion significantly increased during serum deprivation, and was accompanied by increased activation of its receptor, IGF-IR. Additionally, inhibition of IGF-2 activity markedly attenuated the serum deprivation-mediated increase in IGF-IR and Akt activation, HIF-1alpha expression, and also its own transcription, suggesting autocrine regulation of HIF-1alpha expression via IGF-2. Cross-talk between IGF-2/ IGF-IR system and PI3K-Akt pathway was further demonstrated by findings wherein IGF-IR suppression inhibited Akt activation, and IGF-IR activation was inhibited following PI3K inhibition. Furthermore, HIF-1alpha suppression attenuated the serum deprivation-mediated increase in Akt and IGF-IR activation. CONCLUSION: Collectively, our study demonstrates existence of a pro-survival HIF-1alpha-dependent autocrine feedback loop in normoxic, serum-deprived PCa cells.

C/EBPbeta regulates metastatic gene expression and confers TNF-alpha resistance to prostate cancer cells.

BACKGROUND: CCAAT/enhancer-binding protein beta (C/EBPbeta) is a transcription factor and consists of three isoforms, transcription-activating A/B (C/EBPbeta-AB) and transcription inhibitory C (C/EBPbeta-C). We previously reported that C/EBPbeta-C was predominantly expressed in hormone-dependent LNCaP cells, while C/EBPbeta-AB forms were predominant in hormone-independent prostate cancer (HI-PCa) cells. METHODS: Reporter gene analysis was performed to investigate transcriptional activity of C/EBPbeta on metastatic gene expression upon TNF-alpha treatment. NF-kappaB activation and C/EBPbeta protein upregulation were determined by immunoblotting. WST assay was used to determine the role of C/EBPbeta in TNF-alpha-induced cell death. RESULTS: We first determined that the C/EBPbeta-C overexpression or siRNA-mediated C/EBPbeta depletion decreased TNF-alpha-induced promoter activities of Bfl-1, IL-6, and IL-8 genes. IL-6 and IL-8 are autocrine growth factors of HI-PCa cells and Bfl-1 is an anti-apoptotic protein whose function in prostate cancer is yet to be determined. Secondly, we determined differential regulation of C/EBPbeta by TNF-alpha. In DU-145 cells, C/EBPbeta was upregulated by TNF-alpha, but downregulated in LNCaP cells, although NF-kappaB was activated in both cells. This result suggested cell-type specific activation of signaling pathways leading to C/EBPbeta upregulation, which was distinct from that leading to NF-kappaB activation. Most importantly, C/EBPbeta depletion decreased cell growth and sensitized DU-145 cells to TNF-alpha-induced cell death. Conversely, overexpression of C/EBPbeta-A in LNCaP cells increased resistance to TNF-induced cell death and TNF-induced promoter activities of IL-6 and Bfl-1. CONCLUSION: Our study, for the first time, demonstrated that C/EBPbeta regulated cell growth and conferred TNF-alpha resistance to PCa cells, in part, via regulation of metastatic gene expression. Prostate 69: 1435-1447, 2009. (c) 2009 Wiley-Liss, Inc.

Intraoperative dexmedetomidine attenuates stress responses in patients undergoing major spine surgery.

BACKGROUND: Surgical stress induces stress hormone release and sympathetic hyperactivation, resulting in hemodynamic instability. Dexmedetomidine has sympatholytic and hemodynamic stabilizing effects. We investigated whether dexmedetomidine could attenuate stress responses in major spine surgery. METHODS: In this prospective randomized study, 52 patients undergoing spine fusion surgery were randomized to placebo (N.=26) or to dexmedetomidine (N.=26) groups. Dexmedetomidine at a rate of 0.4 µg/kg/h or saline was infused, starting immediately after anesthetic induction and continuing until the end of surgery. Anesthesia was performed using desflurane and remifentanil in both groups. Serum levels of cortisol, epinephrine, norepinephrine, and interleukin-6 were assessed before surgery (T1), at the surgical incision (T2), at the bone procedure (T3), and one hour after surgery (T4). The hemodynamic variables and the autonomic nervous system balance evaluated with heart rate variability were assessed at the same time points. RESULTS: Epinephrine and norepinephrine levels were higher over time in the control rather than in the dexmedetomidine group (P=0.001 and <0.001, respectively). The changes in cortisol, interleukin-6, and hemodynamics were similar between the groups. In the heart rate variability analysis, high-frequency decreased and low-frequency and low-frequency/high-frequency ratio increased during surgery in the control group, whereas they were maintained at the baseline level in the dexmedetomidine group. The changes in high-frequency, low-frequency, and the low-frequency/high-frequency ratio over time differed between the groups (P=0.009, 0.024, and 0.011, respectively). CONCLUSIONS: Intraoperative dexmedetomidine administration reduced stress hormone release and maintained the balance of the autonomic nervous system. Dexmedetomidine could attenuate surgical stress response without untoward hemodynamic adverse events.

Protein phosphatase 1 activation and alternative splicing of Bcl-X and Mcl-1 by EGCG + ibuprofen.

Epigallocatechin-3-gallate (EGCG) and ibuprofen synergistically act to suppress proliferation and enhance apoptosis of prostate cancer cell lines, PC-3 and LNCaP. The purpose of this study was to investigate the mechanism of underlying this synergism. Most interestingly, EGCG + ibuprofen treatment in PC-3 cells resulted in altering the ratio of the splice variants of Bcl-X and Mcl-1, downregulating the mRNA levels of anti-apoptotic Bcl-X(L) and Mcl-1(L) with a concomitant increase in the mRNA levels of pro-apoptotic Bcl-X(s) and Mcl-1(s). However, there were no apparent changes in splicing variants in either ibuprofen or EGCG treated cells. Induction of alternative splicing was correlated with increased activity of protein phosphatase 1 (PP1) in EGCG + ibuprofen-treated cells, since pretreatment with calyculin A and tautomycin blocked EGCG + ibuprofen-induced alternative splicing in PC-3 cells in contrast to pretreatment with okadaic acid. On the other hand, EGCG + ibuprofen treatment in LNCaP cells did not alter splicing variants of Bcl-X and Mcl-1, despite the increase in protein phosphatase activity. In both cell lines, EGCG + ibuprofen inhibited cell proliferation synergistically. Taken together, this study demonstrate for the first time that EGCG + ibuprofen upregulated PP1 activity, which in turn induced alternative splicing of Bcl-X and Mcl-1 in a cell-type specific manner. Our study also demonstrates that the activation of PP1 contributes to the alternative splicing of Mcl-1.

HIF-1 alpha: a key survival factor for serum-deprived prostate cancer cells.

BACKGROUND: Hypoxia-inducible factor-1 alpha (HIF-1 alpha) is commonly overexpressed in prostate cancer (PCa) cells. As PCa cells are known to survive serum deprivation, we investigated the effect of prolonged serum deprivation on HIF-1 alpha expression, and the function of HIF-1 alpha in regulating the survival of normoxic serum-deprived PCa cells. METHODS: HIF-1 alpha protein was assessed by immunoblots. Cell viability and proliferation were assessed by trypan blue assay and flow cytometric analysis. Transcriptional activity was assessed by luciferase reporter assay and RT-PCR. HIF-1 alpha expression was suppressed with siRNA. Activities of HIF-1 alpha-target genes were inhibited with neutralizing antibody. RESULTS: Prolonged serum deprivation is a potent inducer of HIF-1 alpha in PC-3 and LNCaP PCa cells, despite normal oxygen conditions. In contrast, cells grown in the presence of serum did not show HIF-1 alpha protein accumulation. Moreover, HIF-1 alpha protein increase during serum deprivation correlated with increased cell survival, while suppression of HIF-1 alpha expression significantly decreased PCa cell viability. Our results further demonstrate that HIF-1 alpha protein increase is due to increased HIF-1 alpha protein synthesis. First, there was no significant increase in HIF-1 alpha mRNA. Secondly, cycloheximide, a protein synthesis inhibitor, prevented HIF-1 alpha protein increase in serum-deprived PCa cells. Moreover, the expression of HIF-1 alpha-target genes, VEGF and IGF-2, was concomitantly increased in serum-deprived PCa cells, while suppression of HIF-1 alpha expression significantly inhibited their induction. Furthermore, inhibition of IGF-2 activity resulted in a significant decline in PCa cell survival. CONCLUSION: PCa cells counteract the stress of prolonged serum deprivation by upregulating HIF-1 alpha protein which increases IGF-2 expression to promote cell survival.

Translationally regulated C/EBP beta isoform expression upregulates metastatic genes in hormone-independent prostate cancer cells.

BACKGROUND: C/EBP beta is a transcription factor regulating key biological processes including cellular growth and differentiation and its increased expression correlates with tumor invasiveness. Recently, the increased expression of C/EBP beta was reported in proliferative inflammatory atrophy of the prostate, associating with increased COX-2 expression and androgen receptor (AR) downregulation. METHODS: C/EBP beta expression was determined in DU-145, PC-3 and LNCaP cells by immunoblotting. Transient transfection of C/EBP beta expression vectors was performed to investigate translational regulation of its isoform expression. Reporter gene analysis was performed to investigate transcriptional activity of C/EBP beta on metastatic gene expression. RESULTS: We determined that transcriptionally active, full-length C/EBP beta isoforms were dominantly expressed in hormone-independent DU-145 and PC-3 cells, while transcription-repressing truncated isoform was dominant in hormone-dependent LNCaP cells. Our results further showed lack of full-length isoform expression from the transiently transfected C/EBP beta expression vector in LNCaP cells compared to that in PC-3 cells transfected with the same vector, while the expression of truncated isoform was comparable in both cell lines. Interestingly, however, the most upstream initiation site A null mutation restored translation of full-length isoform in LNCaP cells. These results suggest that full-length C/EBP beta isoform expression in LNCaP cells may be suppressed at the upstream initiation sites, likely at site A. Most importantly, C/EBP beta overexpression significantly upregulated promoter activities of IL-8, COX-2, and anti-apoptotic Bfl-1 genes. CONCLUSIONS: Our study demonstrates that C/EBP beta is an important transcription factor upregulating metastatic gene expression and that its isoform expression is differentially regulated at the translational level in prostate cancer cells.

Synergistic cell death by EGCG and ibuprofen in DU-145 prostate cancer cell line.

BACKGROUND: One of the green tea components epigallocatechin-3-gallate (EGCG) significantly prevented the growth of prostate cancer cells. In this study, synergistic effect of EGCG and ibuprofen (EGCG+ibuprofen) was investigated to determine their anti-proliferative and pro-apoptotic action in DU-145 prostate cancer cells. MATERIALS AND METHODS: Cell death analysis, immunoblotting, RT-PCR analysis, and caspase activity assay were used. RESULTS: EGCG+ibuprofen treatment resulted in 90% growth inhibition, while ibuprofen or EGCG alone reduced cell numbers by 25% and 20%, respectively. EGCG+ibuprofen induced MAPK activation, caspase activation and the inhibition of Bfl-1 expression, all of which were blocked by the antioxidant, N-acetyl-L-cysteine (NAC). Moreover, addition of ceramide rescued the NAC-inhibited MAPK activation and pretreatment with the ceramide synthase inhibitor, fumonisin B1, reduced cell death. CONCLUSION: Our results suggest that in DU-145 prostate cancer cells: (i) EGCG+ibuprofen treatment has a synergistic effect on apoptosis, and (ii) oxidative stress, directly or indirectly via ceramide synthesis mediates pro-apoptotic signaling.

Retinoic acid response element in HOXA-7 regulatory region affects the rate, not the formation of anterior boundary expression.

Since it is known that a 307 bp fragment of the position specific regulatory element of human HOXA-7 contains two (DR3 and DR5) retinoic acid response elements (RAREs) at its 3' end, we constructed several deletion constructs containing different numbers of RAREs and examined their effects in vitro and in vivo. The 5' deletion constructs, BM112 and OM213, retaining both RAREs, were highly responsible (about 8 fold induction) for RA in F9 teratocarcinoma cells, versus NM307 (4-5 fold). The construct NS218, with both RAREs deleted but retaining the 5' sequences lost RA responsibility completely, whereas NR271, with one RARE(DR5) deleted retained a 50% inducibility (2.5 fold). In vivo transgenic analysis revealed that the constructs NM307 and NR271, but not OM213 nor BM112, directed the position specific expression pattern. Sequence analysis revealed that HOXA-7 enhancer sequences, including the RARE repeat sequences, were well conserved in human, mouse and chick. Part of the RAREs overlaps with the CDX1 binding site, and sequences of the DR3 RAREs were identical in this species. Two GAGA binding sites were also found to be strictly conserved. Because OM213, which had one GAGA site disrupted but retaining both RAREs, did not direct anterior boundary formation in transgenic mice, these results suggest the importance of the 5' 94 bp region, including the GAGA binding site, in anterior boundary formation and the involvement of the RARE in the rate of expression not in anterior boundary formation.

Different mechanisms of soy isoflavones in cell cycle regulation and inhibition of invasion.

BACKGROUND: Soy isoflavones, genistein, daidzein and glycitein, are thought to have beneficial effects on cancer prevention. MATERIALS AND METHODS: We used cell cycle analysis, invasion assay and immunoblotting to determine the effects of genistein and glycitein on Jurkat T cells. RESULTS: Glycitein inhibited Jurkat cell invasion at a level comparable to the inhibition by genistein. Both genistein and glycitein down-regulated MMP-13 proteolytic activity by 60-70% and MMP-8 expression. Caffeine could block G2/M arrest by genistein, but was unable to block the inhibition of invasion by genistein and glycitein. We also demonstrated that glycitein inhibited proteintyrosine phosphorylation in Jurkat cells. CONCLUSION: We determined, for the first time, that glycitein inhibited Jurkat cell invasion, in part through the down-regulation of MMP-13 activity and MMP-8 expression. Our findings also suggest that soy isoflavones may utilize different mechanisms to exert their effects on cell cycle progression and invasiveness of Jurkat cells.

Targeting the hypoxia inducible factor pathway with mitochondrial uncouplers.

Hypoxia inducible factor-1 (HIF-1) is central to most adaptation responses of tumors to hypoxia, and consists of a hypoxia inducible HIF-1alpha or -2alpha subunit, and a constitutively expressed HIF-1beta subunit. Previously, mitochondrial uncouplers, rottlerin and FCCP, were shown to increase the rate of cellular O(2 )consumption. In this study, we determined that mitochondrial uncouplers, rottlerin and FCCP, significantly decreased hypoxic as well as normoxic HIF-1 transcriptional activity which was in part mediated by down-regulation of the oxygen labile HIF-1alpha and HIF-2alpha protein levels in PC-3 and DU-145 prostate cancer cells. Our results also revealed that mitochondrial uncouplers decreased the expression of HIF target genes, VEGF and VEGF receptor-2. Taken together, our results indicate that functional mitochondria are important in HIF-1alpha and HIF-2alpha protein stability and transcriptional activity during normoxia as well as in hypoxia, and that mitochondrial uncouplers may be useful in the inhibition of HIF pathway in tumors.

Epigallocatechin gallate inhibits HIF-1alpha degradation in prostate cancer cells.

Hypoxia-inducible factor-1, an alphabeta heterodimeric transcription factor, consists of a constitutively expressed HIF-1beta subunit and a hypoxia-inducible HIF-1alpha subunit, and contributes to hypoxia-mediated tumor angiogenesis. Numerous epidemiologic and laboratory studies indicate that green tea has cancer preventive activity which has been attributed to its polyphenol components, the major one being epigallocatechin gallate (EGCG). This study investigated the effect of EGCG on normoxic HIF-1alpha expression in human prostate cancer cells. Surprisingly, we observed an EGCG-induced-dose-dependent increase in HIF-1-mediated transcription and HIF-1alpha protein levels under normoxia. However, concomitant treatment of the prostate cancer cells with EGCG and ferrous ions abolished the increase in HIF-1-mediated transcription that was seen with EGCG treatment alone, suggesting that EGCG may act as a ferrous ion chelator. Furthermore, we determined, for the first time, that EGCG inhibits prolyl hydroxylation of HIF-1alpha, thus preventing HIF-1alpha and pVHL interaction.

Flavonoids inhibit VEGF/bFGF-induced angiogenesis in vitro by inhibiting the matrix-degrading proteases.

Flavonoids have been proposed to act as chemopreventive agents in numerous epidemiological studies and have been shown to inhibit angiogenesis and proliferation of tumor cells and endothelial cells in vitro. Angiogenesis requires tightly controlled extracellular matrix degradation mediated by extracellular proteolytic enzymes including matrix metalloproteinases (MMPs) and serine proteases, in particular, the urokinase-type plasminogen activator (uPA)-plasmin system. In this study, we have investigated the antiangiogenic mechanism of the flavonoids, genistein, apigenin, and 3-hydroxyflavone in a human umbilical vein endothelial cell (HUVEC) model. The stimulation of serum-starved HUVECs with vascular endothelial growth factor/basic fibroblast growth factor (VEGF/bFGF) caused marked increase in MMP-1 production and induced the pro-MMP-2 activation accompanied by the increase in MT1-MMP expression. However, pretreatment with flavonoids before VEGF/bFGF stimulation completely abolished the VEGF/bFGF-stimulated increase in MMP-1 and MT1-MMP expression and pro-MMP-2 activation. Genistein blocked VEGF/bFGF-stimulated increase in TIMP-1 expression and decrease in TIMP-2 expression. Apigenin and 3-hydroxyflavone further decreased TIMP-1 expression below basal level and completely abolished TIMP-2 expression. VEGF and bFGF stimulation also significantly induced uPA expression, most strikingly the level of 33 kDa uPA, and increased the expression of PA inhibitor (PAI)-1. Genistein, apigenin, and 3-hydroxyflavone effectively blocked the generation of 33 kDa uPA, and further decreased the activity of the 55 kDa uPA and the expression of PAI-1 below the basal level. In conclusion, these data suggest that genistein, apigenin, and 3-hydroxyflavone inhibit in vitro angiogenesis, in part via preventing VEGF/bFGF-induced MMP-1 and uPA expression and the activation of pro-MMP-2, and via modulating their inhibitors, TIMP-1 and -2, and PAI-1.