OBJECTIVE

Determine the toxicity, tolerability, and pharmacokinetics of SU5416, a vascular endothelial growth factor receptor-2 (VEGFR-2) tyrosine kinase inhibitor, coadministered with bolus 5-fluorouracil (5-FU), leucovorin, and irinotecan (IFL) in untreated patients with metastatic colorectal cancer.

METHODS

SU5416 (85 or 145 mg/m2) was administered twice weekly throughout a 6-week period along with standard IFL (4 weeks on/2 weeks off). Plasma samples were assayed for SU5416, irinotecan, and SN-38 by reverse-phase HPLC. Contrast enhanced, color Doppler sonography was performed on patients at the MTD to identify changes in tumor perfusion.

RESULTS

Eleven patients received treatment with SU5416 85 mg/m2 (n = 5) or 145 mg/m2 (n = 6). At 85 mg/m2, no DLTs were observed. At 145 mg/m2, grade 3 diarrhea and vomiting were observed during cycle 1; other grade 3 toxicities included fatigue, nausea, anorexia, anemia, pain, urinary retention, and hypertension. The pharmacokinetics of irinotecan and SN-38 were not altered by coadministration of SU5416. SU5416 pharmacokinetics were not altered by IFL. Contrast-enhanced, color Doppler sonography was performed on 2 patients and demonstrated reduced tumor perfusion after treatment in a patient who responded to treatment and increased perfusion in a patient who developed progressive disease. Three patients (27%) had confirmed partial responses, 2 patients (18%) had unconfirmed partial responses, and 4 patients (36%) had stable disease.

CONCLUSIONS

Twice weekly SU5416 can be administered with bolus IFL without unexpected toxicities or altering the pharmacokinetic behavior of the administered drugs. Changes in tumor blood perfusion can be detected by contrast-enhanced, color Doppler sonography. The further development of SU5416 was halted before this study was completed.

OBJECTIVE

To determine the influence of vitreomacular adhesion (VMA) on treatment outcomes in patients with neovascular age-related macular degeneration who were treated with anti-vascular endothelial growth factor agents using a treat and extend treatment regimen.

METHODS

A retrospective consecutive case series of 204 eyes from 181 patients with a minimum of 1 year of follow-up at Wills Eye Hospital Retina Service. Vitreomacular interface characteristics were determined by spectral domain optical coherence tomography. One hundred and fifty-three eyes (75%) had no signs of VMA (non-VMA), and 51 (25%) had VMA.

RESULTS

Baseline mean visual acuity was 20/133 with a mean central retinal thickness of 350.5 μm in the non-VMA group and was 20/145 with 371.8 μm in the VMA group. Mean visual acuity in the non-VMA group was 20/83 and 20/64 at Years 1 and 2, respectively (P < 0.01 to baseline). Mean visual acuity in the VMA group was 20/81 and 20/85 at Years 1 and 2, respectively (P < 0.01 to baseline). The central retinal thickness was 289.71 μm and 267 μm at Years 1 and 2, respectively (P < 0.01 to baseline) in the non-VMA group and was 305.3 μm and 289.24 μm (P < 0.01 to baseline) in the VMA group. The mean total number of injections at Year 1 for non-VMA was 7.4 compared with 8.4 in VMA (P = 0.001) and 5.5 versus 6.7 for the 2 groups in Year 2 (P = 0.027). The mean longest extension at Year 1 was 11.8 weeks compared with 10.1 week (P = 0.005) and for Year 2 was 14.1 weeks compared with 12 weeks (P = 0.041).

CONCLUSIONS

The vitreomacular interface seems to have a significant influence on anti-vascular endothelial growth factor treatment intervals but not visual acuity or exudative control outcomes. Eyes with VMA on spectral domain optical coherence tomography at baseline may require more intensive treatment with decreased ability to extend treatment intervals.

BACKGROUND

Artemisia annua L, artemisinin and artesunate reveal profound activity not only against malaria, but also against cancer in vivo and clinical trials. Longitudinal observations on the efficacy of A. annua in patients are, however missing as of yet.

METHODS

Clinical diagnosis was performed by imaging techniques (MRT, scintigraphy, SPECT/CT) and blood examinations of standard parameters from clinical chemistry. Immunohistochemistry of formalin-fixed, paraffin-embedded tumor material was performed to determine the expression of several biomarkers (cycloxygenase-2 (COX2), epidermal <em>growth</em> <em>factor</em> receptor (EGFR), glutathione S-transferase P1 (GSTP1), Ki-67, MYC, oxidized low density lipoprotein (lectin-like) receptor 1 (LOX1), p53, P-glycoprotein, transferrin receptor (TFR, CD71), <em>vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> (VEGF), von Willebrand <em>factor</em> (CD31)). The immunohistochemical expression has been compared with the microarray-based mRNA expression of these markers in two prostate carcinoma cell lines (PC-3, DU-<em>145</em>).

RESULTS

A patient with prostate carcinoma (pT3bN1M1, Gleason score 8 (4+4)) presented with a prostate specific antigen (PSA) level >800 µg/l. After short-term treatment with bacalitumide (50 mg/d for 14 days) and long-term oral treatment with A. annua capsules (continuously 5 × 50 mg/d), the PSA level dropped down to 0.98 µg/l. MRT, scintigraphy and SPECT/CT verified tumor remission. Seven months later, PSA and ostase levels increased, indicating tumor recurrence and skeletal metastases. Substituting A. annua capsules by artesunate injections (2 × 150 mg twice weekly i.v.) did not prohibit tumor recurrence. PSA and ostase levels rose to 1245 µg/l and 434 U/l, respectively, and MRT revealed progressive skeletal metastases, indicating that the tumor acquired resistance. The high expression of MYC, TFR, and VEGFC in the patient biopsy corresponded with high expression of these markers in the artemisinin-sensitive PC-3 cells compared to artemisinin-resistant DU-<em>145</em> cells.

CONCLUSIONS

Long-term treatment with A. annua capsules combined with short-term bicalitumide treatment resulted in considerable regression of advanced metastasized prostate carcinoma. Controlled clinical trials are required to evaluate the clinical benefit of A. annua in prostate cancer.

OBJECTIVE

Angiogenesis or formation of new blood vessels is an essential process for tumor <em>growth</em>, invasion and metastasis. <em>Vascular</em> <em>Endothelial</em> <em>Growth</em> <em>Factor</em> (VEGF) and its receptors play an important role in angiogenesis-dependent tumors. VEGF-A is the most important <em>factor</em> in angiogenesis process. Human VEGF-A gene consists of eight exons that undergoes alternative exon splicing and produce five different proteins consisting of 121, <em>145</em>, 165, 189 and 206 amino acids (named VEGF121, VEGF<em>145</em>, VEGF165, VEGF189, and VEGF206).

METHODS

In this study, VEGF121 gene synthesized and cloned into the pET-26b plasmid. The recombinant plasmid was transferred into appropriate expression strain of BL-21. Expression of VEGF121 induced by IPTG (Isopropyl β-D-1-thiogalactopyranoside) and confirmed by SDS-PAGE and Western-Blotting. Recombinant VEGF121 was purified by nickel affinity chromatography. HUVECs (Human Umbilical Vein Endothelia Cells) cells were isolated from umbilical vein and the effect of VEGF121 on tube formation of endothelial cells was investigated.

RESULTS

SDS-PAGE and Western-Blotting results verified the purification of VEGF121. The final yield of recombinant protein was about 5mg per liter. Endothelial cell tube formation assay results showed that VEGF121 leads to tube formation of endothelial cell on matrix and induces angiogenesis in vitro.

CONCLUSIONS

Recombinant VEGF121 is important factor in tube formation of endothelial cell, so it could be used in different cancer researches and angiogenesis assay.

<em>Vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> (VEGF) produced by tumor cells plays a central role in stimulating angiogenesis required for solid tumor <em>growth</em>. VEGF-specific antibodies inhibit tumor cell line <em>growth</em> in animal models and a humanized monoclonal anti-VEGF antibody (bevacizumab [Avastin]) is approved as a treatment for metastatic cancer. We hypothesized that administration of an adenoviral (Ad) vector expressing the murine monoclonal antibody equivalent of bevacizumab would suppress human tumor <em>growth</em> in vivo. The Ad vector (AdalphaVEGF) encodes the light chain and heavy chain cDNAs of monoclonal antibody A.4.6.1, a murine antibody that specifically recognizes human VEGF with the same antigen-binding site as bevacizumab. AdalphaVEGF efficacy in vivo was evaluated with A-673 rhabdomyosarcoma and DU <em>145</em> prostate carcinoma cells in human tumor cell xenografts in SCID mice. For both tumor models, AdalphaVEGF directed the expression of high anti-human VEGF IgG antibody titers in vivo, the numbers of mitotic nuclei and blood vessels in the tumor were significantly decreased (p < 0.05), tumor <em>growth</em> was suppressed (p < 0.05), and there was increased survival (p < 0.005). Thus, AdalphaVEGF, encoding a murine monoclonal antibody that is the equivalent of bevacizumab, effectively suppresses the <em>growth</em> of human tumors, suggesting gene therapy as an alternative to bevacizumab monoclonal antibody therapy.

We examined the aberrant microRNA (miRNA) expression profile responsible for the changes in angiogenesis observed in endometriotic lesions. This study revealed characteristic miRNA expression profiles associated with endometriosis in endometrial tissue and endometriotic lesions from the same patient, and their correlation with the most important angiogenic and fibrinolytic <em>factors</em>. miRNA expression was quantified using a microRNA array and reverse-transcription microRNA polymerase chain reaction. Levels of <em>vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> A (VEGFA), epidermal <em>growth</em> <em>factor</em> receptor 2 (EGFR2), phosphatase and tensin homolog (PTEN), and C-X-C chemokine receptor type 4 (CXCR4) were quantified using enzyme-linked immunosorbent assay. The endometrial tissue showed significantly lower levels of miR-200b, miR-15a-5p, miR-19b-1-5p, miR-146a-5p, and miR-200c, and higher levels of miR-16-5p, miR-106b-5p, and miR-<em>145</em>-5p. VEGFA was significantly upregulated, whereas EGFR2, PTEN, and CXCR4 were markedly downregulated, in the endometriotic tissues compared to that in the normal endometrial tissues. In conclusion, differences in the miRNA levels could modulate the expression of VEGFA, EGFR2, PTEN, and CXCR4, and may play an important role in the pathogenesis of endometriosis. The higher angiogenic and proteolytic activities observed in the eutopic endometrium might facilitate the implantation of endometrial cells at ectopic sites.

OBJECTIVE

To evaluate the preclinical pharmacokinetics, antitumor efficacy, and mechanism of action of a novel orally active farnesyltransferase inhibitor, ABT-100.

METHODS

In vitro sensitivity of a panel of human cell lines was determined using proliferation and clonogenic assays. In vivo efficacy of ABT-100 was evaluated in xenograft models (flank or orthotopic) by assessing angiogenesis, proliferation, and apoptosis in correlation with pharmacokinetics. Efficacy of the racemate of ABT-100 (A-367074) was also compared with R115777 (tipifarnib).

RESULTS

ABT-100 inhibited proliferation of cells in vitro carrying oncogenic H-Ras (EJ-1 bladder; IC(50) 2.2 nmol/L), Ki-Ras (DLD-1 colon, MDA-MB-231 breast, HCT-116 colon, and MiaPaCa-2 pancreatic; IC(50) range, 3.8-9.2 nmol/L), and wild-type Ras (PC-3 and DU-<em>145</em>; IC(50), 70 and 818 nmol/L, respectively) as well as clonogenic potential. ABT-100 shows 70% to 80% oral bioavailability in mice. ABT-100 regressed EJ-1 tumors (2-12.5 mg/kg/d s.c., every day for 21 days) and showed significant efficacy in DLD-1, LX-1, MiaPaCa-2, or PC-3 tumor-bearing mice (6.25-50 mg/kg/d s.c. once daily or twice daily orally). A-367074 showed equivalent efficacy to R115777 given at approximately one-fourth the total dose of R115777 for a shorter duration (EJ-1 and LX-1). Antitumor activity was associated with decreased cell proliferation (Ki-67), increased apoptosis (terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling), and decreased angiogenesis. A reduction in tumor angiogenic cytokine levels (<em>vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em>, basic fibroblast <em>growth</em> <em>factor</em>, and interleukin-8) correlated with a reduction in tumor <em>vascular</em>ity (CD31).

CONCLUSIONS

Overall, ABT-100 has an acceptable pharmacokinetic profile, is well tolerated, and possesses broad-spectrum antitumor activity against a series of xenograft models similar to farnesyltransferase inhibitors in clinical development; therefore, it is an attractive candidate for clinical evaluation.

Chronic graft-versus-host disease (cGVHD) is a common late complication of allogeneic stem cell transplantation (allo-SCT). Some cGVHD patients develop skin lesions, and the skin lesions in sclerodermatous cGVHD (s-cGVHD) patients resemble those in progressive systemic sclerosis (PSS), which is characterized by impaired production of circulating <em>endothelial</em> progenitor cells (EPCs). We investigated, retrospectively, whether low EPC production may promote the development of sclerodermatous lesions in cGVHD. Peripheral blood (PB) was obtained from 14 healthy volunteers and 27 allo-SCT patients. Five patients developed s-cGVHD. CD34(+) cells were purified by using the magnetic cell-sorting separation system, and the CD34(+)/CD133(+)/<em>vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> (VEGF) receptor-2(+) EPCs were quantified. The <em>endothelial</em> cell colony-formation potential was evaluated. Serum VEGF and basic fibroblast <em>growth</em> <em>factor</em> (b-FGF) concentrations were measured by ELISA. The s-cGVHD patients had significantly lower median circulating EPCs frequencies than non-s-cGVHD patients or control (<em>145</em> of 20 mL [interquartial range-IQR 107-193] versus 1083.5 [IQR 669.3-2151]; P = .0023, and versus 1530.5 [IQR 961.3-2158]; P = .0012, respectively). They also had impaired median <em>endothelial</em>-forming ability compared to non-s-cGVHD patients or controls (3.8 [IQR 1.0-4.3] versus 12.8 [IQR 8.8-28.8], and versus 26.4 [IQR 23.6-30.6], respectively; P = .0012). Their VEGF and b-FGF serum levels were also higher than in controls. In conclusion, s-cGVHD patients show findings consistent with those seen in PSS with impaired vasculogenesis that may limit blood perfusion and may contribute to the development of sclerodermatous lesions.

Cigarette smoke contains hundreds of potentially toxic compounds and is an important risk <em>factor</em> for cardio<em>vascular</em> disease. However, the key components responsible for <em>endothelial</em> and myocardial dysfunction have not been fully identified. The objective of the present study was to determine the cardio<em>vascular</em> effects of long-term inhalation of carbon monoxide (CO) administrated to give concentrations in the blood similar to those observed in heavy smokers. Female rats were exposed to either CO or air (control group) (n = 12). The CO group was exposed to 200 ppm CO (100 h/wk) for 18 mo. Rats exposed to CO had 24% lower maximal oxygen uptake, longer (<em>145</em> vs. 123 microm) and wider (47 vs. 25 microm) cardiomyocytes, reduced cardiomyocyte fractional shortening (12 vs. 7%), and 26% longer time to 50% re-lengthening than controls. In addition, cardiomyocytes from CO-exposed rats had 48% lower intracellular calcium (Ca2 +) amplitude, 22% longer time to Ca2 + decay, 34% lower capacity of sarcoplasmic reticulum Ca2 +-ATPase (SERCA2a), and 37% less t-tubule area compared to controls. Phosphorylation levels of phospholamban at Ser16 and Thr17 were significantly reduced in the CO group, whereas total concentration of phospholamban and SERCA2a were unchanged. Cardiac atrial natriuretic peptide, <em>vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em>, cyclic guanosine monophosphate, calcineurin, calmodulin, pERK, and pS6 increased, whereas pAkt and pCaMKII delta remained unchanged by CO. <em>Endothelial</em> function and systemic blood pressure were not affected by CO exposure. Long-term CO exposure reduces aerobe capacity and contractile function and leads to pathological hypertrophy. Impaired Ca2 + handling and increased <em>growth</em> <em>factor</em> signaling seem to be responsible for these pathological changes.

OBJECTIVE

To investigate the role of miR-<em>145</em> silencing in the expression of <em>vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> (VEGF), basic fibroblast <em>growth</em> <em>factor</em> (bFGF), B-cell lymphoma-2 (Bcl-2), BCL2-Associated X(bax) and caspase-3 in a<em>vascular</em> necrosis of femoral head (ANFH).

METHODS

A total of 12 healthy wild-types (the control group) and 12 miR-<em>145</em> knock-out (miR-<em>145</em>-/-) (the experimental group) adult New Zealand white rabbits were selected to construct ANFH model with a steroid. Four weeks later, immunohistochemistry, qRT-PCR and Western blot were performed to measure the VEGF, bFGF, Bcl-2, bax, caspase-3, β-catenin as well as c-Myc expression. Terminal-deoxynucleotidyl transferase mediated nick end labeling (TUNEL) staining analysis was used to detect the apoptosis of bone cells in each group.

RESULTS

Compared with the control group, the expression of VEGF, bFGF, Bcl-2, β-catenin and c-Myc in the miR-<em>145</em>-/- group raised (p<0.05). Moreover, the expression level of bax and caspase-3 significantly decreased in the miR-<em>145</em>-/- group (p<0.05). TUNEL staining showed decreased apoptosis in the miR-<em>145</em>-/- group.

CONCLUSIONS

MiR-<em>145</em> silencing promotes bone repair of ANFH via upregulating VEGF, bFGF and inhibiting the bone cells apoptosis through Wnt/β-catenin pathway.

Kawasaki disease (KD) is the most common systemic vasculitis syndrome primarily affecting medium-sized arteries, particularly the coronary arteries. Though KD may be associated with immunological problems, the involvement of microRNAs (miRs) has not been fully described.

We enrolled 23 KD patients and 12 controls. We performed miR and mRNA microarray analysis of peripheral blood mononuclear cells (PBMCs) isolated from acute KD patients and controls. Continuously, we measured specific miRs, mRNA and the expression of proteins by using reverse-transcriptase PCR (RT-PCR) and enzyme-linked immunosorbent assay (ELISA).

We identified strikingly high levels of miR-182 and miR-296-5p during the acute febrile phase, and of miR-93, miR-<em>145</em>-5p, miR-<em>145</em>-3p, and miR-150-3p in the defervescence stage, especially in refractory KD patients. The expression of <em>vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> A (VEGFA) mRNA, previously reported to be controlled by miR-93, was significantly elevated during the febrile phase and normalized upon treatment, negatively correlating with the expression of miR-93. Further, plasma levels of VEGF-A correlated with PBMC VEGFA mRNA expression.

Several miRs are highly specific to the acute phase of KD, and may participate in regulating the expression of genes in pathways associated with KD. In particular, miR-93 may participate in regulating expression of VEGF-A and contribute to the pathogenesis of arteritis in acute KD.

Interleukin 8 (IL-8) and <em>vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> (VEGF) are two cytokines promoting prostate tumor <em>growth</em> and angiogenesis. The main coagulation protease thrombin may modulate the malignant phenotype of prostate cancer cells via its cellular receptor(s). We aimed to investigate the effects of thrombin on IL-8 and VEGF expression in DU <em>145</em> prostate cancer cells. Thrombin induced the expression and secretion of IL-8 and VEGF, with more pronounced effects on IL-8. Target-specific siRNA-induced protease-activated receptor 1 (PAR-1) knockdown completely neutralized thrombin-enhanced cytokine secretion, demonstrating the essential role of PAR-1. Inhibitors of either extracellular signal-regulating kinase (ERK) or phosphatidylinositol 3-kinase (PI3K) partly reversed the thrombin-induced cytokine expression, suggesting that both ERK and PI3K kinase pathways may be involved in IL-8 and VEGF expression. The results suggest that the thrombin/PAR-1 system upregulates cytokines in prostate cancer cells which in turn may contribute to the progression of prostate cancer.

<em>Vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> (VEGF) is a multifunctional cytokine with distinct functions in angiogenesis, lymphangiogenesis, <em>vascular</em> permeability, and hematopoiesis. VEGF is a highly conserved, disulfide-bonded dimeric glycoprotein of 34 to 45 kDa produced by several cell types including fibroblasts, neutrophils, <em>endothelial</em> cells, and peripheral blood mononuclear cells, particularly T lymphocytes and macrophages. Six VEGF isoforms are generated as a result of alternative splicing from a single VEGF gene, consisting of 121, <em>145</em>, 165, 183, 189, or 206 amino acids. VEGF121, VEGF<em>145</em>, and VEGF165 are secreted whereas VEGF183, VEGF189, and VEGF206 are cell membrane-bound. VEGF<em>145</em> has a key role during the <em>vascular</em>ization of the human ovarian follicle and corpus luteum, in the placentation and embryonic periods, and in bone and wound healing, while VEGF165 is the most abundant and biologically active isoform. VEGF has been linked with a number of <em>vascular</em> pathologies including cardio<em>vascular</em> diseases such ischemic heart disease, heart failure, stroke, and diabetes and its related complications. In this review we aimed to present some important roles of VEGF in a number of clinical issues and indicate its involvement in several phenomena from the initial steps of the embryonic period to cardio<em>vascular</em> diseases.

BACKGROUND

<em>Vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> (VEGF) regulates several cell functions including; proliferation, differentiation, permeability, <em>vascular</em> tone, and the production of vasoactive molecules. The purpose of this study was to evaluate the potency of specific short-interfering RNA (siRNA) to suppress human VEGF expression by siRNA and investigate the effects of VEGF down-regulation on the cell proliferation and apoptosis of the human prostate cancer cell lines DU-<em>145</em>.

METHODS

Transfection was performed using X-tremeGENE siRNA transfection reagent. At different time intervals, transfected cells were harvested and total RNA was extracted for RT-PCR. The VEGF content in supernatants were measured by ELISA. Inhibition of cell growth by hVEGF-siRNA was measured by using cell proliferation ELISA BrdU assay. Apoptotic cells were evaluated by using annexin-V-FITC apoptotic detection method.

RESULTS

Transfection of hVEGF-siRNA resulted in statistically significant inhibition of hVEGF-mRNA that in turn caused a marked reduction in the expression of hVEGF. The cell <em>growth</em> was assessed every 24 h for 4 days after siRNA treatment resulted in a marked inhibition of cell proliferation as compared to scramble siRNA. The results of apoptosis showed that approximately 15 % of the cells treated with control-siRNA manifested evident apoptotic changes after 24 hpt, whereas DU-<em>145</em> cells treated with hVEGF-siRNA significantly were positive, that is to say, 53 % at 72 hpt 23.9 ± 2.78 % (P < 0.001) and 13 ± 1.57 % at 96 hpt.

CONCLUSIONS

Our findings indicate that siRNA are effective in eliciting the RNAi pathway in cancerous cells and that specific siRNA efficiently down-regulate VEGF expression. They could decrease VEGF production and induce apoptosis, which may also be linked to the inhibition of cancerous cell proliferation. Therefore, it can be concluded that siRNA-mediated suppression of VEGF represents a powerful tool against prostate cancer cell proliferation. VEGF down-regulation exerts a direct anti-apoptotic function in the DU-<em>145</em> cell lines and promises the development of drugs for cancer therapy.

Previously we developed dicetyl phosphate-tetraethylenepentamine-based polycation liposomes (TEPA-PCL) for use in small interfering RNA (siRNA) therapy. In the present study, mammalian target of rapamycin (mTOR) expression in cancer cells was silenced with mTOR-siRNA (simTOR) formulated in TEPA-PCL modified with Ala-Pro-Arg-Pro-Gly (APRPG), a peptide having affinity for <em>vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> receptor-1 (VEGFR-1). We investigated the effects of inhibition of mTOR, focusing on the differences between cells treated with simTOR and those with rapamycin in terms of Akt (ser473) phosphorylation and antiproliferative effects. Rapamycin treatment is known to induce Akt (ser473) phosphorylation which attenuates the antiproliferative effects of rapamycin. As a result, knockdown of mTOR did not alter or only slightly reduced Akt (ser473) phosphorylation in phosphatase and tensin homolog deleted from chromosome 10 (PTEN)-null (LNCaP and MDA-MB-468 cells) and PTEN-positive (DU <em>145</em> and MDA-MB-231) cells, although rapamycin induced Akt (ser473) phosphorylation of these cells. Rapamycin suppressed the <em>growth</em> of PTEN-null cells, in which the rapamycin-sensitive mTOR complex 1 (mTORC1) is excessively activated. On the other hand, rapamycin did not suppress the <em>growth</em> of PTEN-positive cells possibly through a negative feedback mechanism via the rapamycin-insensitive mTOR complex 2 (mTORC2) signaling pathway. In contrast, simTOR significantly suppressed the <em>growth</em> of cancer cells regardless of the presence of PTEN, possibly through inhibition of both mTORC1 and mTORC2. These results indicate that mTOR knockdown using APRPG-TEPA-PCL/simTOR is likely to be an effective strategy for cancer siRNA therapy.

OBJECTIVE

Oncostatin M (OSM) and interleukin-6 (IL-6) are growth factors for prostate cancer (PC). Vascular endothelial growth factor (VEGF) and urokinase-type plasminogen-activator (u-PA) have been implicated in tumour progression. A possible interaction between IL-6, OSM, u-PA and VEGF in PC was investigated.

METHODS

Primary prostate epithelial cells (PPEC) and DU-145 PC cells were treated with IL-6 or OSM and the effects on u-PA and VEGF expression were studied. Plasma levels of IL-6, OSM, u-PA and VEGF were determined in patients with or without PC.

RESULTS

In DU-145 cells, OSM and IL-6 up-regulated u-PA and VEGF significantly. Higher levels of IL-6 and OSM in metastasising PC than in nonmetastasising PC and benign prostatic hyperplasia (BPH) and correlations between IL-6, OSM, u-PA and VEGF were found.

CONCLUSIONS

OSM and IL-6 increase u-PA and VEGF in DU-145 cells but not in PPEC and possibly, by promoting matrix degradation and angiogenesis, could play a role in the pathogenesis of prostate cancer.

OBJECTIVE

The aim of this study was to evaluate the correlation of protein expressions of CXC chemokine receptor 4 (CXCR4), vascular endothelial growth factor-C (VEGF-C) and cytokeratin 19 (CK-19) with lymph node metastasis (LNM) in patients with hepatocellular carcinoma (HCC), and their survival.

METHODS

The expressions of CXCR4, VEGF-C and CK-19 in HCC patients with (n = 123) or without (n = 145) LNM were determined using tissue microarray and immunohistochemical staining. The relationship between clinicopathological features and CXCR4, VEGF-C and CK-19 were analyzed. Evaluation of immunostaining was performed semiquantitatively by visual assessment.

RESULTS

The UICC T stage, and expressions of nuclear CXCR4, VEGF-C and CK-19 were independent risk factors for LNM. Nuclear CXCR4, VEGF-C and CK-19 expression were predictive factors for LNM in HCC patients. In patients with LNM, the median survival time was 15.1 months for patients with high nuclear CXCR4 expression and 24.5 months for those with low nuclear CXCR4 expression. The median survival time was 15.1 months for patients with high tumor VEGF-C expression and 31.1 months for those with low tumor VEGF-C expression. The median survival time was 12.0 months for patients with positive CK-19 expression and 19.2 months for patients with negative CK-19 expression. Patients with high nuclear CXCR4, VEGF-C or CK-19 expression had significantly poorer prognosis than those with low expression (all P < 0.05). PVT, UICC T stage and expressions of nuclear CXCR4, VEGF-C, and CK-19 were independent prognostic factors.

CONCLUSIONS

Increased protein expressions of nuclear CXCR4, VEGF-C, and CK-19 are independent risk factors for developing lymph node metastasis, and they are significantly correlated with LNM and poor outcome in HCC patients.

BACKGROUND

Angiogenesis plays a crucial role in normal development and carcinogenesis of prostate glands. Placental growth factor (PlGF) belongs to the same family as the vascular endothelial growth factor. The presence of PlGF in human prostate has not been studied. In the current study, we investigated the gene expression profiles of PlGF in human prostate.

METHODS

Gene expression of PlGF-1 and PlGF-2 was assessed by RT-PCR and direct sequencing. Gene expression levels were quantified by real-time PCR, and we compared the transcript levels among benign prostate hyperplasia (BPH), prostate caner (CAP) and CAP after androgen deprivation therapy (CAP-Tx).

RESULTS

Human prostate cancer cells, LNCaP, PC-3 and DU-145, expressed both PlGF-1 and -2 mRNAs. Human prostate tissues, BPH, CAP and CAP-Tx, also expressed both types of PlGF mRNA. BPH and CAP-Tx expressed similar levels of PlGF, however, CAP expressed significantly lower levels of PlGF than BPH or CAP-Tx (p < 0.01).

CONCLUSIONS

PlGF mRNA was expressed in human prostate. Significantly lower levels of PlGF in CAP in comparison with those in BPH or CAP-Tx suggested that PlGF might have effects on vasculogenesis and angiogenesis in prostate disease.

In human and ovine fetuses, glucocorticoids stimulate leptin secretion, although the extent to which leptin mediates the maturational effects of glucocorticoids on pulmonary development is unclear. This study investigated the effects of leptin administration on indices of lung structure and function before birth. Chronically catheterized singleton sheep fetuses were infused iv for 5 days with either saline or recombinant ovine leptin (0.5 mg/kg · d leptin (LEP), 0.5 LEP or 1.0 mg/kg · d, 1.0 LEP) from 125 days of gestation (term ∼<em>145</em> d). Over the infusion, leptin administration increased plasma leptin, but not cortisol, concentrations. On the fifth day of infusion, 0.5 LEP reduced alveolar wall thickness and increased the volume at closing pressure of the pressure-volume deflation curve, interalveolar septal elastin content, secondary septal crest density, and the mRNA abundance of the leptin receptor (Ob-R) and surfactant protein (SP) B. Neither treatment influenced static lung compliance, maximal lung volume at 40 cmH2O, lung compartment volumes, alveolar surface area, pulmonary glycogen, protein content of the long form signaling Ob-Rb or phosphorylated signal transducers and activators of transcription-3, or mRNA levels of SP-A, C, or D, elastin, <em>vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em>-A, the <em>vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> receptor 2, angiotensin-converting enzyme, peroxisome proliferator-activated receptor γ, or parathyroid hormone-related peptide. Leptin administration in the ovine fetus during late gestation promotes aspects of lung maturation, including up-regulation of SP-B.

<em>Vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> (VEGF) and its receptors are known to play an important role in normal and pathological hematopoiesis but the prognostic impact of VEGF isoform transcripts in acute myeloid leukemia (AML) has not been addressed. We conducted a single-institution prospective study to analyze the impact of these angiogenic <em>factors</em> and the expression of their receptors on the survival of adult patients newly diagnosed with AML. We investigated the levels of VEGF transcript isoforms VEGF121, -<em>145</em>, -165, -189 and -206 and their receptors, VEGFR-1 and VEGFR-2, using quantitative reverse transcriptase polymerase chain reaction assays in peripheral blood mononuclear cells (PBMCs) of 67 consecutive AML patients at diagnosis. VEGF total protein was measured for comparison with mRNA levels in PBMCs. The VEGF121 splice variant transcript in AML PBMCs was significantly higher than in the normal controls. VEGF transcripts were quantified in all samples while its protein was detected in 42/67 (63%) of AML samples. High levels of VEGF121, VEGF165 transcripts and VEGF protein in AML were significantly related to a worse prognosis when analyzing overall survival (p<0.0001, p=0.019 and p=0.012, respectively) or event-free survival (p<0.0001, p=0.010 and p=0.047) using univariate analysis. In multivariable analysis only VEGF121 expression remained an independent prognostic <em>factor</em> for either event-free survival or overall survival [aHR=8.83 (3.48-22.4), p<0.0001, and aHR=9.52 (3.41-26.6), p<0.0001]. No prognostic value was observed for the other isoforms and the two receptors. Our findings show that the level of VEGF121 mRNA in circulating cells from AML patients is a strong independent prognostic parameter, which could be useful in the management of unselected AML patients.

To test the hypothesis that coronary flow and coronary flow reserve are developmentally regulated, we used fluorescent microspheres to investigate the effects of acute (6 h) pulmonary artery banding (PAB) on baseline and adenosine-enhanced right (RV) and left ventricular (LV) blood flow in two groups of twin ovine fetuses (100 and 128 days of gestation, term <em>145</em> days, n = 6 fetuses/group). Within each group, one fetus underwent PAB to constrict the main pulmonary artery diameter by 50%, and the other twin served as a nonbanded control. Physiological measurements were made 6 h after the surgery was completed; tissues were then harvested for analysis of selected genes that may be involved in the early phase of coronary <em>vascular</em> remodeling. Within each age group, arterial blood gas values, heart rate, and mean arterial blood pressure were similar between control and PAB fetuses. Baseline endocardial blood flow in both ventricles was greater in 100 than 128-day fetuses (RV: 341 +/- 20 vs. 230 +/- 17 ml*min(-1)*100 g(-1); LV: 258 +/- 18 vs. 172 +/- 23 ml*min(-1)*100 g(-1), both P < 0.05). In both age groups, RV and LV endocardial blood flows increased significantly in control animals during adenosine infusion and were greater in PAB compared with control fetuses. After PAB, adenosine further increased RV blood flow in 128-day fetuses (from 416 +/- 30 to 598 +/- 33 ml*min(-1)*g(-1), P < 0.05) but did not enhance blood flow in 100-day animals (490 +/- 59 to 545 +/- 42 ml*min(-1)*100 g(-1), P>> 0.2). RV <em>vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> and Flk-1 mRNA levels were increased relative to controls (P < 0.05) in 128 but not 100-day PAB fetuses. We conclude that in the ovine fetus, developmentally related differences exist in 1) baseline myocardial blood flows, 2) the adaptive response of myocardial blood flow to acute systolic pressure load, and 3) the responses of selected genes involved in vasculogenesis to increased load in the fetal myocardium.

OBJECTIVE

Thymosin (Tβ4) may have various biological effects that are relevant to the pathogenesis of rheumatoid arthritis (RA). This study was performed to gain insight into the relevance of Tβ4 in the pathogenesis of inflammatory arthritis.

METHODS

The level of Tβ4 in synovial fluid from patients with osteoarthritis (OA) or RA was measured by enzyme-linked immunosorbent assay. An association between Tβ4 and matrix metalloproteinase (MMP)-1 and MMP-13 (collagenases), MMP-2 and MMP-9 (gelatinases), MMP-7, adiponectin, lactoferrin, vascular endothelial growth factor (VEGF), urokinase-type plasminogen activator (uPA), interleukin (IL)-6, IL-8 and prostaglandin E2 (PGE(2) ) in synovial joint fluids from OA and RA patients were investigated.

RESULTS

The level of Tβ4 in the synovial joint fluid of patients with OA and RA was (mean ± SD) 145 ± 88 and 1359 ± 1685 ng/mL, respectively. The level of Tβ4 in the synovial joint fluid of RA patients was significantly associated with the levels of MMP-9, MMP-13, VEGF, uPA, IL-6 and IL-8, but not with MMP-1, MMP-2, MMP-7, adiponectin and lactoferrin. In contrast, the level of Tβ4 in the synovial joint fluid of patients with OA was not associated with any of these molecules.

CONCLUSIONS

The results suggest that Tβ4 may play an important role in bone degradation and inflammation in RA but not OA, although nothing is known about the molecular mechanisms mediating Tβ4 in arthritic joints. The role of Tβ4 in arthritis should be studied to understand its relevance to the pathogenic processes in arthritis.

Puerarin, a major isoflavonoid compound from Chinese herb Kudzu roots, has been widely used for the treatment of hypertensive and cardio<em>vascular</em> diseases in China. Here, we investigated puerarin's beneficial effects on the cardio<em>vascular</em> system in angiotensin (Ang) II-induced hypertensive rats. Sprague-Dawley rats were treated with Ang II for 5 days or with puerarin for 10 days followed by Ang II and puerarin for 5 days. Endothelium-dependent relaxation (EDR) to acetylcholine was determined using an organ chamber bath. Ang II increased the systolic blood pressure (SBP: 178 ± 5 mmHg vs. 112 ± 3 mmHg in control, p < 0.05), aortic (30%, p < 0.05), and left ventricular (LV) weight (23%); puerarin reduced SBP (160 ± 2 mmHg, p < 0.05), aortic, and left ventricular weight in Ang II-infused rats. Puerarin also reduced aortic medial thickness and myocardial cell surface area in Ang II-infused rats. Compared with control rats, Ang II infused rats exhibited an impaired EDR with reduction in the protein expression of phosphor-eNOS at Ser 1177 and an increase in the expression of gp91phox (85%), p22phox (113%), transforming <em>growth</em> <em>factor</em> β1 (<em>145</em>%) and <em>vascular</em> cell adhesion molecule 1 (82%). Puerarin improved EDR and reversed the changes in Ang II-induced protein expression of above molecules. Our results demonstrate that in Ang II-induced hypertensive rats, puerarin protects against <em>endothelial</em> dysfunction and end organ damage with a mild reduction in SBP, and that the cardio<em>vascular</em> beneficial effects of puerarin may be in part attributed to its anti-oxidant and upregulation of phosphor-eNOS.

OBJECTIVE

Retrospective evaluation of high-dose-rate brachytherapy (HDR BT) in early oral cancer and factors influencing tumor control.

METHODS

A total of 30 patients with T1-T3N0 tongue and floor of mouth cancer were treated with tumor excision±elective neck dissection and HDR BT 18×3 Gy b.i.d. The Kaplan-Meier model was used for survival analyses, and the log-rank test and Cox regression analyses were used to evaluate the influence of T-stage, histologic grade, resection margin, depth of invasion, and vascular endothelial growth factor (VEGF) intensity on local control (LC), nodal control (NC), disease-free survival (DFS), and overall survival (OS). Median followup was 40 months (6-145).

RESULTS

Actuarial 3-year LC, NC, DFS, DFS after salvage treatment, and OS were 85.4%, 69.2%, 65.4%, 75.6%, and 73.0%, respectively. The log-rank test and univariate Cox regression analysis revealed the following correlations, namely tumor grade correlated with LC, DFS, and OS; T-stage with NC and DFS; depth of invasion and VEGF intensity with NC, DFS, and OS. Associations detected on the multivariate analysis were as follows: tumor grade with LC, depth of invasion with NC, depth of invasion and tumor grade with DFS, and VEGF intensity with DFS after salvage treatment. Only one case of osteoradionecrosis and two cases of soft tissue necrosis occurred.

UNASSIGNED

The HDR BT 18×3 Gy b.i.d. is a safe treatment of early oral cancer with a good LC. The T-stage, tumor grade, depth of invasion, and intensity of VEGF were significant predictors of locoregional control.