Clinical trials of VEGF receptor tyrosine kinase inhibitors in pancreatic cancer

Pancreatic cancer is the fourth leading cause of cancer-related deaths in Western countries and is among the deadliest diseases in humans. At present, gemcitabine is the standard chemotherapy for advanced pancreatic cancer, although (despite its use) prognosis continues to be dismal with a median survival of < 6 months. While targeting tumor vasculature has provided improved outcomes in colon, lung, breast and renal cell cancers, trials of angiogenesis inhibitors have lagged behind in pancreatic cancer. This review provides the rationale for exploring antiangiogenic therapies in the treatment of pancreatic cancer as well as summarizes present clinical development of VEGF receptor tyrosine kinase inhibitors and their application to pancreatic cancer.     

Clinical trials of VEGF receptor tyrosine kinase inhibitors in pancreatic cancer

Pancreatic cancer is the fourth leading cause of cancer-related deaths in Western countries and is among the deadliest diseases in humans. At present, gemcitabine is the standard chemotherapy for advanced pancreatic cancer, although (despite its use) prognosis continues to be dismal with a median survival of < 6 months. While targeting tumor vasculature has provided improved outcomes in colon, lung, breast and renal cell cancers, trials of angiogenesis inhibitors have lagged behind in pancreatic cancer. This review provides the rationale for exploring antiangiogenic therapies in the treatment of pancreatic cancer as well as summarizes present clinical development of VEGF receptor tyrosine kinase inhibitors and their application to pancreatic cancer.     

Protein kinase C-mediated Ca2+ entry in HEK 293 cells transiently expressing human TRPV4

1. We investigated whether protein kinase C (PKC) activation stimulates Ca2+ entry in HEK 293 cells transfected with human TRPV4 cDNA and loaded with fura-2. 2. Phorbol 12-myristate 13-acetate (PMA), a PKC-activating phorbol ester, increased the intracellular Ca2+ concentration ([Ca2+]i) in a dose-dependent manner, with an EC50 value of 11.7 nm. Exposure to a hypotonic solution (HTS) after PMA further increased [Ca2+]i. Two other PKC-activating phorbol esters, phorbol 12,13-didecanoate (PDD) and phorbol 12,13-dibutyrate, also caused [Ca2+]i to increase. 3. The inactive isomer 4alpha-PMA was less effective and the peak [Ca2+]i increase was significantly smaller than that induced by PMA. In contrast, 4alpha-PDD produced a monophasic or biphasic [Ca2+]i increase with a different latency, while 4alpha-phorbol had no effect. 4. The PMA-induced [Ca2+]i increase was abolished by prior exposure to bisindolylmaleimide (BIM), a PKC-specific inhibitor, and suppressed by the nonspecific PKC inhibitor 1-(5-isoquinolinesulphonyl)-2-methylpiperazine. The [Ca2+]i increase induced by 4alpha-PMA, 4alpha-PDD or HTS was not significantly affected by BIM. 5. These results suggest that both PKC-dependent and -independent mechanisms are involved in the phorbol ester-induced activation of TRPV4, and the PKC-independent pathway is predominant in HTS-induced Ca2+ entry.     

Design and structure-activity relationship of a new class of potent VEGF receptor tyrosine kinase inhibitors

A series of substituted 4-anilinoquinazolines and related compounds were synthesized as potential inhibitors of vascular endothelial growth factor (VEGF) receptor (Flt and KDR) tyrosine kinase activity. Enzyme screening indicated that a narrow structure-activity relationship (SAR) existed for the bicyclic ring system, with quinazolines, quinolines, and cinnolines having activity and with quinazolines and quinolines generally being preferred. Substitution of the aniline was investigated and clearly indicated that small lipophilic substituents such as halogens or methyl were preferred at the C-4' position. Small substituents such as hydrogen and fluorine are preferred at the C-2' position. Introduction of a hydroxyl group at the meta position of the aniline produced the most potent inhibitors of Flt and KDR tyrosine kinases activity with IC(50) values in the nanomolar range (e.g. 10, 12, 13, 16, and 18). Investigation of the quinazoline C-6 and C-7 positions indicates that a large range of substituents are tolerated at C-7, whereas variation at the C-6 is more restricted. At C-7, neutral, basic, and heteroaromatic side chains led to very potent compounds, as illustrated by the methoxyethoxy derivative 13 (IC(50) < 2 nM). Our inhibitors proved to be very selective inhibitors of Flt and KDR tyrosine kinase activity when compared to that associated with the FGF receptor (50- to 3800-fold). Observed enzyme profiles translated well with respect to potency and selectivity for inhibition of growth factor stimulated proliferation of human umbilical vein endothelial cells (HUVECs). Oral administration of selected compounds to mice produced total plasma levels 6 h after dosing of between 3 and 49 microM. In vivo efficacy was demonstrated in a rat uterine oedema assay where significant activity was achieved at 60 mg/kg with the meta hydroxy anilinoquinazoline 10. Inhibition of growth of human tumors in athymic mice has also been demonstrated: compound 34 inhibited the growth of established Calu-6 lung carcinoma xenograft by 75% (P < 0.001, one tailed t-test) following daily oral administration of 100 mg/kg for 21 days.     

Inhibition of the VEGF receptor system with tyrosine kinase inhibitors. Angiogenesis inhibition in oncology

Die Tyrosinkinase-Inhibitoren mit antiangiogenen Eigenschaften, die sie als Angiogenese-Inhibitoren qualifizieren, haben ihre Zulassung als Monotherapeutikum zurzeit bei Nischentumoren, bei denen es keine guten Therapiealternativen gab, in Placebo-kontrollierten Studien erreicht: GIST, RCC und HCC. Progressionsfreie Zeitintervalle und Gesamtüberlebenszeiten k?nnen um einige Monate verl?ngert werden. Die spannende Frage ist jedoch, welche antiangiogenen Tyrosinkinase-Inhibitoren sich auch bei den Massentumoren wie Kolon-, Mamma-, Bronchial- und Prostatakarzinom etablieren k?nnen. Zahlreiche Studien sind auch für Sunitinib (n=157) und Sorafenib (n=172) unter www.clinicaltrials.gov gelistet. Potentiell sind Multikinase-Inhibitoren mit antiangiogenen Eigenschaften in der Lage, eindrucksvolle therapeutische Effekte im Sinne von Tumorregressionen zu induzieren, weil sie neben der Wirkung auf die Endothelzelle auch direkt auf die Tumorzelle wirken. Multikinase-Inhibition bedeutet allerdings auch St?rung von multiplen Signaltransduktionswegen und damit assoziiert auch multiple Nebenwirkungsspektren.Das VEGF/VEGFR-System ist ohne Zweifel wichtig, es ist jedoch nicht monokausal zu betrachten. Andere "Sub"-Systeme der Angiogenese sind bedeutsam, vielleicht sogar wichtiger als bisher erkannt. Erst wenn die komplizierte Regulation der Angio-/Vaskulogenese wirklich verstanden wird, k?nnte es m?glich werden, die Tumorangiogenese so zu inhibieren, dass metastasierte Tumorerkrankungen zu einer wirklich chronischen Erkrankung konvertiert werden k?nnen. Essentiell für weiteren Fortschritt ist weitere intensive Grundlagenforschung auf dem Gebiet der Tumorangiogenese. Wichtig für die Anwendung sehr teurer neuer Medikamente wird ferner die Identifizierung der Patienten sein, bei denen der Einsatz dieser Medikamente mit einem lang anhaltenden klinischen Nutzen verbunden ist (eine Pr?selektion). Welche pr?diktiven Tests hierfür nutzbar sein werden ist zurzeit v?llig offen. Einen angiogenen Ph?notyp durch einige (wenige) Biomarker zu definieren, ist bisher nicht gelungen. Auch dieses Forschungsgebiet steckt erst am Anfang und wird zur Festlegung und Steuerung einer komplexen Tyrosinkinase-Inhibitionstherapie in Zukunft sehr wichtig werden, denn die "number needed to treat" (NNT), um einen sinnvollen Therapieeffekt zu induzieren, sollte idealerweise klein sein. Heute ist diese NNT noch viel zu gross und damit ?konomisch betrachtet für das Gesundheitssystem ungünstig, für die Pharmaindustrie jedoch günstig, weil Umsatz generiert wird.Der aktuelle Stand der Labor- und klinischen Angiogeneseforschung kann in drei sehr aktuellen Büchern nachgelesen werden. Wie Wachstumsfaktoren, ihre Rezeptoren und Signaltransduktion und Onkogene und Krebs zusammenh?ngen, ist in einer aktuellen Ubersicht anschaulich dargestellt. Die Wechselwirkungen sind hochinteressant und sehr komplex. Die spezifische Angiogenese-Inhibition ist ein neues therapeutisches Konzept in der medizinischen Onkologie mit einer grossen Perspektive. Aus der von J. Folkman 1971 erstmals beschriebenen Hypothese, dass Angiogenese und Tumorwachstum fest verbunden sind, ist heute klinische Anwendbarkeit mit Angiogenese-Inhibitoren in der Onkologie geworden und damit liegt man sicherlich nicht falsch zu konstatieren, das dies der Beginn einer neuen Ara ist.     

血管生成素1与血管内皮生长因子165对人胃癌细胞增殖与凋亡的影响

目的研究人血管生成素1(Ang-1)、血管内皮生长因子165(VEGF165)以及两种因子相互作用对人胃癌细胞株MGC-803增殖与凋亡的影响。方法应用MTT法测定腺病毒绿色荧光蛋白(Ad-GFP)(B组)、Ad-Ang-1(C组)、Ad-VEGF165(D组)以及Ad-Ang-1+Ad-VEGF165/2(E组)对MGC-803细胞增殖的影响;另设对照组(A组)。采用流式细胞术分析血清饥饿时其对凋亡的影响;运用Western blot方法检测Bcl-2和Bax蛋白的表达。结果 24、48、72 h时,C、D、E组吸光度均明显高于B、A组(P<0.05);E组吸光度明显高于C、D组(P<0.05)。与A组或B组相比,C、D、E组均能够抑制细胞凋亡(P<0.01),其中E组抑制凋亡作用最强。C、D、E组Bcl-2蛋白表达均比B、A组明显增高(P<0.05),Bax蛋白的表达则明显降低(P<0.05)。结论 Ang-1、VEGF165和Ang-1+VEGF165/2能够明显促进MGC-803细胞体外增殖,抑制血清饥饿时的凋亡;Ang-1与VEGF165联合作用要显著强于单用Ang-1或VEGF165。     

Bi-directional transport of GABA in human embryonic kidney (HEK-293) cells stably expressing the rat GABA transporter GAT-1.

1. Bi-directional GABA-transport was studied by performing uptake and superfusion experiments in human embryonic kidney 293 cells stably expressing the rat GABA transporter rGAT-1. 2. K(M) and V(max) values for [(3)H]-GABA uptake were 11.7+/-1.8 microM and 403+/-55 pmol min(-1) 10(-6) cells (n=9), respectively. 3. Kinetic analysis of outward transport was performed by pre-labelling the cells with increasing concentrations of [(3)H]-GABA and triggering outward transport with 333 microM GABA. Approximate apparent K(M) and V(max) values were 12 mM and 50 pmol min(-1) 10(-6) cells, respectively. 4. GABA re-uptake inhibitors (RI; e.g. tiagabine), as well as, substrates of the rGAT-1 (e.g. GABA, nipecotic acid) concentration dependently decreased [(3)H]-GABA uptake and increased efflux of [(3)H]-GABA from pre-labelled cells. The IC(50) values for inhibiting uptake and the EC(50) values for increasing efflux were significantly correlated (r(2)=0.99). 5. On superfusion, RI antagonized the efflux-enhancing effect of the substrates. The effect of the latter was markedly augmented in the presence of ouabain (100 microM), whereas the effect of RI remained unchanged. The most likely explanation for the release enhancing effect of RI is interruption of ongoing re-uptake. 6. The structural GABA-analogue 2,4-diamino-n-butyric acid (DABA) exhibited a bell-shaped concentration response curve on [(3)H]-GABA efflux with the maximum at 1 mM, and displayed a deviation from the sigmoidal inhibition curve in uptake experiments in the same concentration range. At concentrations below 1 mM, DABA inhibited [(3)H]-GABA uptake non-competitively, while at 1 mM and above the inhibition of uptake followed a competitive manner. 7. The results provide information of GABA inward and outward transport, and document a complex interaction of the rGAT-1 with its substrate DABA.     

Pharmacology of human sulphonylurea receptor SUR1 and inward rectifier K(+) channel Kir6.2 combination expressed in HEK-293 cells

1. The pharmacological properties of K(ATP) channels generated by stable co-expression of the sulphonylurea receptor SUR1 and the inwardly rectifying K(+) channel Kir6.2 were characterized in HEK-293 cells. 2. [(3)H]-Glyburide (glibenclamide) bound to transfected cells with a B(max) value of 18.5 pmol mg(-1) protein and with a K(D) value of 0.7 nM. Specific binding was displaced by a series of sulphonylurea analogues with rank order potencies consistent with those observed in pancreatic RINm5F insulinoma and in the brain. 3. Functional activity of K(ATP) channels was assessed by whole cell patch clamp, cation efflux and membrane potential measurements. Whole cell currents were detected in transfected cells upon depletion of internal ATP or by exposure to 500 microM diazoxide. The currents showed weak inward rectification and were sensitive to inhibition by glyburide (IC(50)=0.92 nM). 4. Metabolic inhibition by 2-deoxyglucose and oligomycin treatment triggered (86)Rb(+) efflux from transfected cells that was sensitive to inhibition by glyburide (IC(50)=3.6 nM). 5. Diazoxide, but not levcromakalim, evoked concentration-dependen decreases in DiBAC(4)(3) fluorescence responses with an EC(50) value of 14.1 microM which were attenuated by the addition of glyburide. Diazoxide-evoked responses were inhibited by various sulphonylurea analogues with rank order potencies that correlated well with their binding affinities. 6. In summary, results from ligand binding and functional assays demonstrate that the pharmacological properties of SUR1 and Kir6.2 channels co-expressed in HEK-293 cells resemble those typical of native K(ATP) channels described in pancreatic and neuronal tissues.     

Effects of protein tyrosine kinase inhibitors on cytokine-induced adhesion molecule expression by human umbilical vein endothelial cells.

1. Endothelial cells can be stimulated by the pro-inflammatory cytokines interleukin (IL)-1 alpha and tumour necrosis factor (TNF) alpha to express the leukocyte adhesion molecules E-selectin, vascular cell adhesion molecule (VCAM)-1 and intercellular adhesion molecule (ICAM)-1 but the intracellular signalling mechanisms leading to this expression are incompletely understood. We have investigated the role of protein tyrosine kinases (PTK) in adhesion molecule expression by cytokine-activated human umbilical vein endothelial cells (HUVEC) using the PTK inhibitors genistein and herbimycin A, and the protein tyrosine phosphatase (PTP) inhibitor sodium orthovanadate. 2. Maximal E-selectin expression induced by incubation of HUVEC for 4 h with IL-1 alpha (100 u ml-1) and TNF alpha (100 u ml-1) was dose-dependently inhibited by genistein and herbimycin A. Although similar effects were seen on phorbol 12-myristate, 13-acetate (PMA)-induced expression, this was not due to inhibition of protein kinase C (PKC) activity as the selective inhibitors of PKC, bisindolylmaleimide (BIM), Ro31-7549 or Ro31-8220 did not affect IL-1 alpha- or TNF alpha-induced E-selectin expression at concentrations which maximally inhibited PMA-induced expression. 3. Genistein inhibited VCAM-1 expression induced by incubation of HUVEC for 24 h with TNF alpha or IL-1 alpha whereas it did not affect ICAM-1 expression induced by 24 h incubation with either of these cytokines. Herbimycin A inhibited both VCAM-1 and ICAM-1 expression induced by TNF alpha. 4. Basal expression of E-selectin, VCAM-1 and ICAM-1 was dose-dependently enhanced by sodium orthovanadate. In contrast, vanadate differentially affected TNF alpha-induced expression of these molecules with maximal E-selectin and ICAM-1 expression being slightly enhanced and VCAM-1 expression dose-dependently reduced. 5. We also studied the effects of PTK and PTP inhibitors on adhesion of the human pre-myeloid cell line U937 to TNF alpha-stimulated HUVEC. Adhesion of U937 cells to HUVEC pretreated for 4 or 24 h with TNF alpha was dose-dependently inhibited by genistein and herbimycin A but unaffected by daidzein. Adhesion of U937 cells after 4 h was partially inhibited by blocking antibodies against both E-selectin and VCAM-1 but after 24 h was only inhibited by anti-VCAM-1. 6. Sodium orthovanadate had no effect on TNF alpha-induced U937 adhesion but dose-dependently enhanced adhesion to unstimulated HUVEC. Vanadate-induced adhesion was inhibited by an antibody against VCAM-1. 7. These results demonstrate that PTK-mediated phosphorylation events are important for the regulation of adhesion molecule expression by human endothelial cells, and additionally show that PTK inhibitors differentially affect upregulation of different adhesion molecules, implicating divergent regulatory pathways for cytokine-induced adhesion molecule expression.     

The effect of five artificial sweeteners on Caco-2, HT-29 and HEK-293 cells

Context: Artificial sweeteners (AS) have been associated with tumor development (including colon cancer) in both animals and humans although evidence has been conflicting. Objectives: Additional research was thus conducted by studying the effects of 5 AS on the morphology, cell proliferation and DNA in cells by utilizing Caco-2, HT-29 (colon) and HEK-293 (kidney) cell lines. Materials and methods: Cells were exposed to sodium cyclamate, sodium saccharin, sucralose and acesulfame-K (0–50?mM) and aspartame (0–35?mM) over 24, 48 and 72 hours. Morphological changes were presented photographically and % cell viability was determined by using the MTT cell viability assay. Possible DNA damage (comet assay) induced by the AS (0.1, 1 and 10?mM, treated for 24, 48 and 72 hours) was studied. The appearance of “comets” was scored from no damage to severe damage (0–4). Results: Cells became flatter and less well defined at higher AS concentrations (>10?mM). At concentrations >10?mM, decreased cell viability was noted with both increasing concentration and increasing incubation time for all cell lines tested. In general, HEK-293 cells seemed to be less affected then the colon cancer cells. Sucralose and sodium saccharin seemed to elicit the greatest degree of DNA fragmentation of all the sweeteners tested in all the cell lines used. Discussion: Morphological cell alterations, cell viability and DNA fragmentation seemed to be more in the colon cancer cells. Conclusions: Further studies have to be performed to clarify mechanisms involved causing these alterations in mammalian cells.     

Effects of Na+ channel blocker,pilsicainide, on HERG current expressed in HEK-293 cells

PURPOSE: Pilsicainide, classified as a relatively pure Na+ channel blocker, occasionally causes QT prolongation, suggesting inhibitory actions on K+ currents. We studied effects of pilsicainide on the K+ channel current of the human ether-a-go-go-related gene (HERG) in heterologous expression system. METHODS: The Patch-clamp technique in whole-cell configuration was used to record the channel current of HERG stably expressed in HEK293 cells. RESULTS: Pilsicainide suppressed peak currents of HERG channel during depolarizing pulses and tail currents upon repolarization. Pilsicainide blocked HERG current with IC50 = 20.4 microM and Hill coefficient = 0.98. Voltage-dependent activation was shifted in a negative direction by approximately 10 mV by 10 to 20 microM pilsicainide. Block increased with depolarization to voltages between -20 and 0 mV and reached the maximum level at positive voltages to 0 mV without further increase. Following drug equilibration for 10 minutes (holding potential at -100 mV), the peak outward current upon the first depolarization showed time-dependent block; tail current block was maximal. Frequency-dependent block evaluated from tail current was absent with pulse frequencies of 1.33, 0.5, and 0.2 Hz. After a steady state block was achieved, time course of current activation and deactivation was slowed by pilsicainide, and steady-state inactivation and time course of fast inactivation were mildly affected. CONCLUSIONS: Pilsicainide blocks HERG current with a preferential affinity, at least, to the open state of the channels with a fast access to binding sites.     

Effects of genistein and herbimycin, tyrosine kinase inhibitors, on catecholamine release in bovine adrenal chromaffin cells

1 The effects of genistein and herbimycin, tyrosine kinase inhibitors, on catecholamine (CA) release were examined in bovine adrenal chromaffin cells. 2 In intact cells, genistein (10-100 microm) and herbimycin (3-30 microm) inhibited CA release induced by acetylcholine (ACh; 100 microm) or the nicotinic receptor stimulant 1,1-dimethyl-4-phenyl-piperazinium (DMPP; 10 microm), but did not affect CA release induced by high K+ (40 mm). 3 Genistein and herbimycin inhibited (45)Ca2+ uptake induced by ACh (100 microm). 4 Neither genistein nor herbimycin affected [(3)H]nicotine binding with nicotinic receptors. 5 In beta-escin-permeabilized cells, neither genistein nor herbimycin affected CA release induced by Ca2+ (1 microm). 6 These results suggest that protein tyrosine kinase plays the facilitatory role in the regulation of CA release induced by nicotinic receptor stimulation in stimulus-secretion coupling of bovine adrenal chromaffin cells.     

Effects of immunoglobulin D on expression of IgD receptor and protein tyrosine kinase signaling in human CD4~+ T cells

OBJECTIVE To observe whether human CD4~+T cells could be activated by immuno-globulin D(IgD) via IgD receptor(IgDR)-Lck.METHODS Human CD4~+T cells were purified from peripheral blood mononuclear cells(PBMCs) with microbeads.The viability of T cells were detected by CCK-8.The binding affinity and expression of IgDR on T cells were detected by flow cytometry.The protein expression of IgDR,Lck and P-Lck were analyzed by western blot.RESULTS IgD could concentration-dependent bind to IgDR on CD4~+T cells.The expression of IgDR was increased in response to treatment with IgD in a time-dependent and concentration-dependent manner.Stimulating by IgD resulted in enhanced phosphorylation of Lck compared with that in the medium control sample.The expression of Lck was not changed.As inhibitor of PTK,Herbimycin A or A770041,which combined with IgD could significantly inhibit phosphorylation of Lck(Tyr~(394)).The proliferation promoting effect of IgD was blocked by Herbimycin A or A770041.IgD could stimulate CD4~+T cell activation and proliferation through upregulating activating tyrosine residue of Lck(Tyr~(394)) phosphorylation.CONCLUSION These results demonstrate that IgD exaggerates CD4~+T cell activities,which may be through promoting Lck phosphorylation.     

Epidermal growth factor receptor tyrosine kinase regulates the human inward rectifier potassium K(IR)2.3 channel, stably expressed in HEK 293 cells

BACKGROUND AND PURPOSE

The detailed molecular modulation of inward rectifier potassium channels (including the K_IR2.3 channel) is not fully understood. The present study was designed to determine whether human K_IR2.3 (K_IR2.3) channels were regulated by protein tyrosine kinases (PTKs).

EXPERIMENTAL APPROACH

Whole-cell patch voltage-clamp, immunoprecipitation, Western blot analysis and site-directed mutagenesis were employed to determine the potential PTK phosphorylation of Kir2.3 current in HEK 293 cells stably expressing Kir2.3 gene.

KEY RESULTS

The broad-spectrum PTK inhibitor genistein (10 µM) and the selective epidermal growth factor (EGF) kinase inhibitor AG556 (10 µM) reversibly decreased K_IR2.3 current and the effect was reversed by the protein tyrosine phosphatase inhibitor, orthovanadate (1 mM). Although EGF (100 ng·mL⁻¹) and orthovanadate enhanced K_IR2.3 current, this effect was antagonized by AG556. However, the Src-family tyrosine kinase inhibitor PP2 (10 µM) did not inhibit K_IR2.3 current. Tyrosine phosphorylation of K_IR2.3 channels was decreased by genistein or AG556, and was increased by EGF or orthovanadate. The decrease of tyrosine phosphorylation of K_IR2.3 channels by genistein or AG556 was reversed by orthovanadate or EGF. Interestingly, the response of K_IR2.3 channels to EGF or AG556 was lost in the K_IR2.3 Y234A mutant channel.

CONCLUSION AND IMPLICATIONS

These results demonstrate that the EGF receptor tyrosine kinase up-regulates the K_IR2.3 channel via phosphorylation of the Y234 residue of the WT protein. This effect may be involved in the endogenous regulation of cellular electrical activity.     

Effects of luteolin and quercetin, inhibitors of tyrosine kinase, on cell growth and metastasis-associated properties in A431 cells overexpressing epidermal growth factor receptor

1. Flavonoids display a wide range of pharmacological properties including anti-inflammatory. Anti-mutagenic, anti-carcinogenic and anti-cancer effects. Here, we evaluated the effects of eight flavonoids on the tumour cell proliferation, cellular protein phosphorylation, and matrix metalloproteinase (MMPs) secretion. 2. Of the flavonoids examined, luteolin (Lu) and quercetin (Qu) were the two most potent agents, and significantly inhibited A431 cell proliferation with IC50 values of 19 and 21 micronM, respectively. 3. The epidermal growth factor (EGF) (10 nM) promoted growth of A431 cells (+25+/-4.6%) and mediated epidermal growth factor receptor (EGFR) tyrosine kinase activity and autophosphorylation of EGFR were inhibited by Lu and Qu. At concentration of 20 micronM, both Lu and Qu markedly decreased the levels of phosphorylation of A431 cellular proteins, including EGFR. 4. A431 cells treated with Lu or Qu exhibited protuberant cytoplasmic blebs and progressive shrinkage morphology. Lu and Qu also time-dependently induced the appearance of a ladder pattern of DNA fragmentation, and this effect was abolished by EGF treatment. 5. The addition of EGF only marginally diminished the inhibitory effect of luteolin and quercetin on the growth rate of A431 cells, treatment of cellular proteins with EGF and luteolin or quercetin greatly reduced protein phosphorylation, indicating Lu and Qu may act effectively to inhibit a wide range of protein kinases, including EGFR tyrosine kinase. 6. EGF increased the levels of matrix metalloproteinase-2 (MMP-2) and matrix metalloproteinase-9 (MMP-9), while Lu and Qu appeared to suppress the secretion of these two MMPs in A431 cells. 7. Examination of the relationship between the chemical structure and inhibitory effects of eight flavonoids reveal that the double bond between C2 and C3 in ring C and the OH groups on C3' and C4' in ring B are critical for the biological activities. 8. This study demonstrates that the inhibitory effects of Lu and Qu, and the stimulatory effects of EGF, on tumour cell proliferation, cellular protein phosphorylation, and MMP secretion may be mediated at least partly through EGFR. This study supports the idea that Lu and Qu may have potential as anti-cancer and anti-metastasis agents.     

Effects of exogenous female sex-steroid hormones on lymphocyte β2-adrenoceptors in normal females

We have previously shown that lymphocyte β₂-adrenoceptors (AR) are under cyclical control of sex-steroid hormones with greater receptor density during the luteal phase of the menstrual cycle. It has also been postulated that abnormal cyclical regulation of β₂-AR might be a possible mechanism for premenstrual asthma. The effects of exogenous female sex-steroid hormones on lymphocyte β₂-AR function were studied in eight normal healthy females. They were evaluated at two successive menstrual cycles, during the follicular phase (day 1–6). They were randomized to receive single oral doses of either ethinyloestradiol 50 μg or medroxyprogesterone 10 mg in a cross-over study. Lymphocyte β₂-AR parameters were evaluated at baseline (t₀), 24 h (t₂₄) and 72 h (t₇₂) after ingestion. Baseline levels of progesterone and oestradiol were comparable on both cycles. Receptor density (B_max) increased significantly (P<0.01) from t₀ after progesterone but not oestradiol at t₂₄: a 1.39-fold geometric mean difference (95% CI 0.96–2.00) between t₂₄vs t₀. Receptor affinity (K_d) and maximal cAMP response to isoprenaline (E_max) were not altered by either treatment. These results show that exogenous progesterone but not oestradiol, given during the follicular phase, significantly increased β₂-AR. This, therefore, suggests that endogenous progesterone is probably responsible for previously observed increase in B_max during the luteal phase of the female menstrual cycle. These findings may suggest possible therapeutic strategies for modulation of β₂-AR in premenstrual asthma.     

Effects of serum on C3b-stimulated release of prostaglandins and thromboxane B2 from human monocytes

Previous studies have demonstrated that C3b stimulates the release of prostaglandin E and thromboxane B2 from human monocytes in serum-free cultures. We have examined the influence of serum on this phenomenon, and have found that addition of normal human serum or fetal calf serum to such cultures at concentrations of 0.1-5.0% results in enhanced release of both eicosanoids in C3b-stimulated cultures, while release in control cultures is unaffected or, in many cases, inhibited. Addition of human serum albumin or transferrin to serum-free cultures is not sufficient to mimic the serum effect. Fetal calf and normal human sera increase the efficacy of C3b in stimulating release of both prostaglandin E and thromboxane B2 at all but the lowest doses tested, but fail to significantly alter the kinetics or release or the acquired unresponsiveness of monocytes to C3b that occurs following culture periods of greater than 24 h. By preincubation of purified C3b in normal human serum, it can be shown that such serum degrades C3b stimulatory activity, but at a rate that is slow enough to permit expression of its activity when added at the beginning of the culture period. In contrast, the stimulatory activity of E. coli lipopolysaccharide is unaffected by pretreatment with serum. Thus, in contrast to the inhibitory effect of serum on the activity of C3 fragments in in vitro assays of certain mononuclear cell functions such as lymphocyte transformation and antibody production, serum enhances the ability of C3b to stimulate monocyte prostaglandin release.     

A combinatorial in silico and cellular approach to identify a new class of compounds that target VEGFR2 receptor tyrosine kinase activity and angiogenesis

BACKGROUND AND PURPOSE

Vascular endothelial growth factor receptor 2 (VEGFR2) is an attractive therapeutic target for the treatment of diseases such as cancer. Small-molecule VEGFR2 inhibitors of a variety of chemical classes are currently under development or in clinical use. In this study, we describe the de novo design of a new generation pyrazole-based molecule (JK-P3) that targets VEGFR2 kinase activity and angiogenesis.

EXPERIMENTAL APPROACH

JK-P compound series were designed using de novo structure-based identification methods. Compounds were tested in an in vitro VEGFR2 kinase assay. Using primary endothelial cells, JK-P compounds were assessed for their ability to inhibit VEGF-A-stimulated VEGFR2 activation and intracellular signalling. We tested these compounds in cell migration, proliferation and angiogenesis assays.

KEY RESULTS

JK-P3 and JK-P5 were predicted to bind the VEGFR2 kinase domain with high affinity, and both compounds showed pronounced inhibition of endogenous VEGFR2 kinase activity in primary human endothelial cells. Only JK-P3 inhibited VEGF-A-stimulated VEGFR2 activation and intracellular signalling. Interestingly, JK-P3 inhibited endothelial monolayer wound closure and angiogenesis but not endothelial cell proliferation. Both compounds inhibited fibroblast growth factor receptor kinase activity in vitro, but not basic fibroblast growth factor-mediated signalling in endothelial cells.

CONCLUSIONS AND IMPLICATIONS

This is the first report that describes an anti-angiogenic inhibitor based on such a pyrazole core. Using a de novo structure-based identification approach is an attractive method to aid such drug discovery. These results thus provide an important basis for the development of multi-tyrosine kinase inhibitors for clinical use in the near future.     

Knock-down of argonaute 2 (AGO2) induces apoptosis in myeloid leukaemia cells and inhibits siRNA-mediated silencing of transfected oncogenes in HEK-293 cells

Understanding the role of oncomirs allows new insights into the development of modern therapeutic approaches for the repression of multiple oncomirs in cancer cells. At present, no suitable approach is available to repress the development of multiple oncomirs in cancer cells. Herein, we report that argonaute 2 (AGO2) could be a unique molecule to regulate the development of multiple oncomirs in cancer cells. Knock-down of AGO2 by custom-made AGO2 siRNA resulted in the induction of apoptosis in myeloid leukaemia cells (HL-60). Further investigations revealed that knock-down of AGO2 by custom-made AGO2 siRNA in HEK-293 cells resulted in silencing of the expression of target genes vascular endothelial growth factor A and histone deacetylase 2, which are known to be involved in the development of myeloid leukaemia. From these results, it can be predicted that AGO2 could regulate siRNA-mediated RNAi pathways in cancer cells. Furthermore, we investigated the possible implication of AGO2 in drug-induced apoptosis. Investigations revealed that treatment with the newly synthesized drug analogue SH-03[{(7S,7aR,13aS)-9,10-dimethoxy-3,3-dimethyl-7,7a,13,13atetrahydro-3H-chromeno[3,4-b]pyrano[2,3-h]chromen-7-ol}] could induce AGO2-mediated apoptosis in myeloid leukaemia cells via intrinsic apoptotic pathways independent of Dicer.