Efficacy of LGD1069 (Targretin), a retinoid X receptor-selective ligand, for treatment of uterine leiomyoma

The conventional treatment of uterine leiomyomas, or fibroids, with gonadotropin-releasing hormone (GnRH) agonists is often associated with serious side effects, necessitating short-term, palliative use of this therapy. Therefore, we examined a retinoid X receptor (RXR)-selective ligand, LGD1069, as a possible treatment for leiomyoma. LGD1069 has demonstrated efficacy as a chemopreventive agent in the N-nitroso-N-methylurea (NMU)-induced rat mammary carcinoma model and is a therapeutic agent in several epithelial tumor models. Previous studies have shown that it has both antitumor effects and antiestrogenic activity in the rat uterus, suggesting the potential utility of this agent for treatment of hormonally dependent uterine fibroids. The expression of retinoid receptors in tumors and cell lines derived from leiomyomas arising in the Eker rat was confirmed by Northern analysis. After treatment for 4 months with LGD1069, the number of grossly observable tumors was substantially reduced although the total incidence of tumors, including microscopic lesions, remained unaffected, suggesting an effect of the compound on tumor growth kinetics rather than on tumor initiation. Analysis of terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) staining and determination of 5-bromo-2-deoxyuridine (BrdU) incorporation indicated that the reduction in grossly observable tumors that occurred in treated animals was mediated by a significant increase in the level of apoptosis rather than a decrease in cell proliferation. These results suggest that LGD1069 may be an effective therapeutic agent for uterine leiomyoma that may inhibit tumor growth and, consequently, alleviate the symptoms associated with this disease.     

Agonist, antagonist, and inverse agonist characteristics of TIPP (H-Tyr-Tic-Phe-Phe-OH), a selective delta-opioid receptor ligand

Recent evidence indicates that the well established delta-opioid antagonist TIPP (H-Tyr-Tic-Phe-Phe-OH) also displays agonist activity in several cellular models. Therefore, it is possible that TIPP, and structurally related compounds, might represent a novel class of opioid agonists exhibiting unique characteristics. The purpose of this study was to examine the properties of TIPP at selected points of the signal transduction pathway (i.e., receptor binding, G-protein activation, and effector regulation) in GH(3)DORT cells (GH(3) cells expressing delta-opioid receptors) and compare them with that of an established delta-opioid agonist, [D-Pen(2),D-Pen(5)]-enkephalin (DPDPE). DPDPE exhibited properties of an agonist in all assays. In contrast, TIPP demonstrated characteristics of an agonist, antagonist, or inverse agonist, depending on the step in the signal transduction cascade examined and the assay conditions employed. In receptor binding assays, the addition of guanine nucleotides and sodium ions increased the affinity of TIPP for delta-opioid receptors in both membrane preparations and digitonin-permeabilized cells, which is characteristic of an inverse agonist. In assays measuring G-protein activation, TIPP failed to stimulate guanosine 5'-O-(3-[(35)S]thio)triphosphate ([(35)S]GTPgammaS) binding in membrane preparations, which is consistent with an antagonist profile. However, when using cells semi-permeabilized with digitonin, TIPP exhibited properties of an agonist, producing concentration-dependent, antagonist-reversible stimulation of [(35)S]GTPgammaS binding. Finally, in assays examining regulation of the intracellular effector adenylyl cyclase, TIPP exhibited characteristics of an agonist, producing inhibition of enzyme activity in both membrane preparations and whole cells. Therefore, although DPDPE and TIPP act similarly as agonists to regulate the intracellular effector adenylyl cyclase, they demonstrate significant differences in the signal transduction cascade preceding this final point of convergence.     

6-(7-Nitro-2,1,3-benzoxadiazol-4-ylthio)hexanol, a specific glutathione S-transferase inhibitor, overcomes the multidrug resistance (MDR)-associated protein 1-mediated MDR in small cell lung cancer

In the present work, we have investigated the antitumor activity of 6-(7-nitro-2,1,3-benzoxadiazol-4-ylthio)hexanol (NBDHEX) on aggressive small cell lung cancer. NBDHEX not only is cytotoxic toward the parental small cell lung cancer H69 cell line (LC(50) of 2.3 +/- 0.6 micromol/L) but also overcomes the multidrug resistance of its variant, H69AR, which overexpresses the ATP-binding cassette transporter multidrug resistance-associated protein 1 (MRP1; LC(50) of 4.5 +/- 0.9 micromol/L). Drug efflux experiments, done in the presence of a specific inhibitor of MRP1, confirmed that NBDHEX is not a substrate for this export pump. Interestingly, NBDHEX triggers two different types of cell death: a caspase-dependent apoptosis in the H69AR cells and a necrotic phenotype in the parental H69 cells. The apoptotic pathway triggered by NBDHEX in H69AR cells is associated with c-Jun NH(2)-terminal kinase and c-Jun activation, whereas glutathione oxidation and activation of p38(MAPK) is observed in the NBDHEX-treated H69 cells. In contrast to the parental cells, the higher propensity to die through apoptosis of the H69AR cell line may be related to the lower expression of the antiapoptotic protein Bcl-2. Therefore, down-regulation of a factor crucial for cell survival makes H69AR cells more sensitive to the cytotoxic action of NBDHEX, which is not a MRP1 substrate. We have previously shown that NBDHEX is cytotoxic toward P-glycoprotein-overexpressing tumor cell lines. Therefore, NBDHEX seems a very promising compound in the search for new molecules able to overcome the ATP-binding cassette family of proteins, one of the major mechanisms of multidrug resistance in cancer cells.     

The effects of fenoldopam, a selective dopamine receptor agonist, on systemic and renal hemodynamics in normotensive subjects.

OBJECTIVE: Acute renal failure, frequently a consequence of renal vasoconstriction and subsequent renal ischemia, is a common problem for which no proven preventive or therapeutic agents exist. Fenoldopam is a new, selective, dopamine-1 receptor agonist that causes both systemic and renal arteriolar vasodilation. In hypertensive patients, fenoldopam rapidly decreases blood pressure, increases renal blood flow, and maintains or improves the glomerular filtration rate. We sought to determine a dose of fenoldopam that increases renal blood flow without inducing hypotension in normotensive patients and to explore the role of volume status (sodium replete vs. deplete) in these effects. DESIGN: Randomized, double-blind, placebo-controlled, cross-over study. SETTING: Clinical research unit. PATIENTS: Fourteen normal male volunteers. INTERVENTIONS: Renal plasma flow (para-aminohippurate clearance) and glomerular filtration rate (inulin clearance) were measured during three fixed, escalating doses of fenoldopam (0.03, 0.1, and 0.3 Lg/kg/min) on both a high-sodium and a low-sodium diet. MEASUREMENTS AND MAIN RESULTS: Fenoldopam significantly increased renal plasma flow in a dose-dependent manner compared with placebo: 670 + 148 vs. 576 + 85 mUmin at 0.03 iLg/kg/min; 777 + 172 vs. 579 + 80 mUmin at 0.1 tig/kg/min; and 784 + 170 vs. 592 + 165 mUmin at 0.3 ilg/kg/min (p < .05 fenoldopam vs. placebo at all three doses). Glomerular filtration rate was maintained. At the lowest dose (i.e., 0.03 ILg/kg/min), significant renal blood flow increases occurred without changes in systemic blood pressure or heart rate. At 0.1 and 0.3 Lgl/kg/ min, systolic blood pressure did not change, but diastolic blood pressure was slightly lower in the fenoldopam group than in the placebo group: 62.5 + 6.4 vs. 63.6 + 2.6 mm Hg, respectively, at 0.3 tg/kg/min (p < .05). None of the effects of fenoldopam were altered by volume status. CONCLUSIONS: Fenoldopam increased renal blood flow in a dose-dependent manner compared with placebo, and, at the lowest dose, significantly increased renal blood flow occurred without changes in systemic blood pressure or heart rate. These findings will be useful in designing future studies exploring the role of fenoldopam in preventing or treating renal failure in patients who are not hypertensive.     

Reversal of multidrug resistance by small interfering RNA (siRNA) in doxorubicin-resistant MCF-7 breast cancer cells

Purpose

Resistance to anticancer drugs is a serious obstacle to cancer chemotherapy. A common form of multidrug resistance (MDR) is caused by the overexpression of transmembrane transporter proteins P-glycoprotein (P-gp) and multidrug resistance-associated protein-1 (MRP1), encoded by MDR1 and MRP1 genes, respectively. These proteins lead to reduced intracellular drug concentration and decreased cytotoxicity by means of their ability to pump the drugs out of the cells. Breast cancer tumor resistance is mainly associated with overexpression of P-gp/MDR1. Although some chemical MDR modulators aim to overcome MDR by interfering functioning of P-gp, their toxicities limit their usage in clinics. Consequently, RNA interference mediated sequence specific inhibition of the expression of P-gp/MDR1 mRNA may be an efficient tool to reverse MDR phenotype and increase the success of chemotherapy. Aim of this study was resensitizing doxorubicin-resistant breast cancer cells to anticancer agent doxorubicin by selective downregulation of P-gp/MDR1 mRNA.

Methods

The effect of the selected MDR1 siRNA, and MRP1 expression after MDR1 silencing was determined by qPCR analysis. Intracellular drug accumulation and localization was investigated by confocal laser scanning microscopy after treatment with MDR1 siRNA. XTT cell proliferation assay was performed to determine the effect of MDR1 silencing on doxorubicin sensitivity.

Results

The results demonstrated that approximately 90% gene silencing occurred by the selected siRNA targeting MDR1 mRNA. However, the level of MRP1 mRNA did not change after MDR1 downregulation. Silencing of P-gp encoding MDR1 gene resulted in almost complete restoration of the intracellular doxorubicin accumulation and relocalization of the drug in the nuclei. Introduction of siRNA resulted in about 70% resensitization to doxorubicin.

Conclusions

Selected siRNA duplex was shown to effectively inhibit MDR1 gene expression, restore doxorubicin accumulation and localization, and enhance chemosensitivity of resistant cells, which makes it a suitable candidate for therapeutic applications.     

Rifaximin,a pregnane X receptor (PXR) activator regulates apoptosis in a murine model of breast cancer

The present study was proposed to investigate the effect of rifaximin (RFX) on methyl nitrosourea (MNU) induced mammary gland carcinoma in albino wistar rats. Animals were randomized and divided among four groups of six animals each. Group I (control 0.9% normal saline, 3 ml kg⁻¹, p.o.); Group II (toxic control, MNU 47 mg kg⁻¹, i.v.); Group III (RFX, 25 mg kg⁻¹, p.o.); Group IV (RFX, 50 mg kg⁻¹, p.o.). Toxicity was induced by single i.v. injection of MNU. MNU treatment was evident with increased alveolar bud count, differentiation score, up-regulated inflammatory enzyme markers (COX, LOX, NO and H₂S) and oxidative stress markers (TBAR''s, protein carbonyl, SOD, catalase and Ach). The mammary gland surface architecture was studied using SEM, carmine staining and H&E staining. The treatment with RFX elicited noticeable restoration of the overall histological architecture in the experimental animals similar to the control. In the MNU treated toxic group, the levels of oxidative stress markers significantly increased in comparison to the control, which was subsequently restored after RFX treatment. Furthermore, RFX up regulated the levels of caspase 3 and caspase 8, when compared to the MNU treated animals. MNU associated toxicity was also ascertained, when determined for UCHL-1, COX, NF-κBp65, BAD, and BCL-xl expression, while RFX demonstrated modulation of the same.

The present study was proposed to investigate the effect of rifaximin (RFX) on methyl nitrosourea (MNU) induced mammary gland carcinoma in albino wistar rats.     

(R,S)-4-phosphonophenylglycine, a potent and selective group III metabotropic glutamate receptor agonist, is anticonvulsive and neuroprotective in vivo.

Group III metabotropic glutamate receptors (mGluRs) are thought to modulate neurotoxicity of excitatory amino acids, via mechanisms of presynaptic inhibition, such as regulation of neurotransmitter release. Here, we describe (R,S)-4-phosphonophenylglycine (PPG) as a novel, potent, and selective agonist for group III mGluRs. In recombinant cell lines expressing the human receptors hmGluR4a, hmGluR6, hmGluR7b, or hmGluR8a, EC50 values for (R,S)-PPG of 5.2 +/- 0.7 microM, 4.7 +/- 0.9 microM, 185 +/- 42 microM, and 0.2 +/- 0.1 microM, respectively, were measured. The compound showed EC50 and IC50 values of >/=200 microM at group I and II hmGluRs and was inactive at cloned human N-methyl-D-aspartate, alpha-amino-3-hydroxy-5-methyl-isoxazole-4-propionate, and kainate receptors (>300 microM). On the other hand, it showed micromolar affinity for a Ca2+/Cl--dependent L-glutamate binding site in rat brain, similar to other phosphono-substituted amino acids like L-2-amino-4-phosphonobutyrate. In cultured cortical neurons, (R, S)-PPG provided protection against a toxic pulse of N-methyl-D-aspartate (EC50 = 12 microM), which was reversed by the group III mGluR antagonist (R,S)-alpha-methylserine-O-phosphate but not by the group II antagonist (2S)-alpha-ethylglutamate. Moreover, (R,S)-PPG protected against N-methyl-D-aspartate- and quinolinic acid-induced striatal lesions in rats and was anticonvulsive in the maximal electroshock model in mice. In contrast to the group III mGluR agonists L-2-amino-4-phosphonobutyrate and L-serine-O-phosphate, (R,S)-PPG showed no proconvulsive effects (2200 nmol i.c.v.). These data provide novel in vivo evidence for group III mGluRs as attractive targets for neuroprotective and anticonvulsive therapy. Also, (R,S)-PPG represents an attractive tool to analyze the roles of group III mGluRs in nervous system physiology and pathology.     

Moxonidine, a selective alpha2-adrenergic and imidazoline receptor agonist, produces spinal antinociception in mice.

alpha2-Adrenergic receptor (AR)-selective compounds produce antihypertensive and antinociceptive effects. Moxonidine alleviates hypertension in multiple species, including humans. This study demonstrates that intrathecally administered moxonidine produces antinociception in mice. Antinociception was detected via the (52.5 degrees C) tail-flick and Substance P (SP) nociceptive tests. Moxonidine was intrathecally administered to ICR, mixed C57BL/6 x 129/Sv [wild type (WT)], or C57BL/6 x 129/Sv mice with dysfunctional alpha2aARs (D79N-alpha2a). The alpha2AR-selective antagonist SK&F 86466 and the mixed I1/alpha2AR-selective antagonist efaroxan were tested for inhibition of moxonidine-induced antinociception. Moxonidine prolonged tail-flick latencies in ICR (ED50 = 0.5 nmol; 0. 3-0.7), WT (0.17 nmol; 0.09-0.32), and D79N-alpha2a (0.32 nmol; 0. 074-1.6) mice. Moxonidine inhibited SP-elicited behavior in ICR (0. 04 nmol; 0.03-0.07), WT (0.4 nmol; 0.3-0.5), and D79N-alpha2a (1.1 nmol; 0.7-1.7) mice. Clonidine produced antinociception in WT but not D79N-alpha2a mice. SK&F 86466 and efaroxan both antagonized moxonidine-induced inhibition of SP-elicited behavior in all mouse lines. SK&F 86466 antagonism of moxonidine-induced antinociception implicates the participation of alpha2ARs. The comparable moxonidine potency between D79N-alpha2a and WT mice suggests that receptors other than alpha2a mediate moxonidine-induced antinociception. Conversely, absence of clonidine efficacy in D79N-alpha2a mice implies that alpha2aAR activation enables clonidine-induced antinociception. When clinically administered, moxonidine induces fewer side effects relative to clonidine; moxonidine-induced antinociception appears to involve a different alpha2AR subtype than clonidine-induced antinociception. Therefore, moxonidine may prove to be an effective treatment for pain with an improved side effect profile.     

Moxonidine, a selective imidazoline/alpha(2) adrenergic receptor agonist, synergizes with morphine and deltorphin II to inhibit substance P-induced behavior in mice

The alpha(2) adrenergic receptor (AR) class of catecholamine/imidazoline (I) agonists, such as norepinephrine and clonidine, produce spinal antinociceptive synergy when co-administered with opioids. We have observed that intrathecally administered moxonidine, a selective I(1)/alpha(2) (AR) agonist, produces antinociception. The present experiments tested moxonidine for ability to synergize with morphine, deltorphin II, and DAMGO (Tyr-D-Ala-NMe-Phe-Gly(ol)) to inhibit substance P-elicited nociceptive behavior in Institute of Cancer Research mice. Moxonidine, morphine, deltorphin II, and DAMGO inhibited substance P-elicited nociceptive behavior with full efficacy. Effective dose 50% (ED(50)) values were calculated and equi-effective dose ratios of the combinations moxonidine-morphine, moxonidine-deltorphin II, and moxonidine-DAMGO were determined. The interactions were tested by isobolographic analysis. The observed ED(50) values of the combinations were statistically compared against their respective calculated theoretical additive ED(50) values. The combinations of moxonidine-morphine and moxonidine-deltorphin II resulted in significant leftward shifts in the dose-response curves compared to those of each agonist administered separately. The ED(50) values of the dose-response curves of these combinations were significantly less than the corresponding calculated theoretical additive ED(50) values; these results indicated that moxonidine synergizes with both morphine and deltorphin II. In contrast, combining moxonidine with DAMGO did not increase the potencies of the agonists (in combination) when compared to the potencies of each agonist administered separately. These results indicated that the moxonidine-DAMGO interaction is subadditive. Collectively, these data demonstrate that moxonidine combined with some opioid agonists produces spinal antinociceptive synergy. Spinally administered moxonidine-opioid combinations may prove an effective therapeutic strategy to manage pain.     

Lack of the purinergic receptor P2X(7) results in resistance to contact hypersensitivity

Sensitization to contact allergens requires activation of the innate immune system by endogenous danger signals. However, the mechanisms through which contact allergens activate innate signaling pathways are incompletely understood. In this study, we demonstrate that mice lacking the adenosine triphosphate (ATP) receptor P2X(7) are resistant to contact hypersensitivity (CHS). P2X(7)-deficient dendritic cells fail to induce sensitization to contact allergens and do not release IL-1β in response to lipopolysaccharide (LPS) and ATP. These defects are restored by pretreatment with LPS and alum in an NLRP3- and ASC-dependent manner. Whereas pretreatment of wild-type mice with P2X(7) antagonists, the ATP-degrading enzyme apyrase or IL-1 receptor antagonist, prevents CHS, IL-1β injection restores CHS in P2X(7)-deficient mice. Thus, P2X(7) is a crucial receptor for extracellular ATP released in skin in response to contact allergens. The lack of P2X(7) triggering prevents IL-1β release, which is an essential step in the sensitization process. Interference with P2X(7) signaling may be a promising strategy for the prevention of allergic contact dermatitis.     

The multidrug resistance protein 5 (ABCC5) confers resistance to 5-fluorouracil and transports its monophosphorylated metabolites

5'-Fluorouracil (5-FU), used in the treatment of colon and breast cancers, is converted intracellularly to 5'-fluoro-2'-deoxyuridine (5-FUdR) by thymidine phosphorylase and is subsequently phosphorylated by thymidine kinase to 5'-fluoro-2'-dUMP (5-FdUMP). This active metabolite, along with the reduced folate cofactor, 5,10-methylenetetrahydrofolate, forms a stable inhibitory complex with thymidylate synthase that blocks cellular growth. The present study shows that the ATP-dependent multidrug resistance protein-5 (MRP5, ABCC5) confers resistance to 5-FU by transporting the monophosphate metabolites. MRP5- and vector-transfected human embryonic kidney (HEK) cells were employed in these studies. In 3-day cytotoxicity assays, MRP5-transfected cells were approximately 9-fold resistant to 5-FU and 6-thioguanine. Studies with inside-out membrane vesicles prepared from transfected cells showed that MRP5 mediates ATP-dependent transport of 5 micromol/L [(3)H]5-FdUMP, [(3)H]5-FUMP, [(3)H]dUMP, and not [(3)H]5-FUdR, or [(3)H]5-FU. The ATP-dependent transport of 5-FdUMP showed saturation with increasing concentrations and had a K(m) of 1.1 mmol/L and V(max) of 439 pmol/min/mg protein. Uptake of 250 micromol/L 5-FdUMP was inhibited by dUMP, cyclic nucleotide, cyclic guanosine 3',5'-monophosphate, amphiphilic anions such as probenecid, MK571, the phosphodiesterase inhibitors, trequinsin, zaprinast, and sildenafil, and by the chloride channel blockers, 5-nitro-2-(3-phenylpropylamino)-benzoic acid and glybenclamide. Furthermore, the 5-FU drug sensitivity of HEK-MRP5 cells was partially modulated to that of the HEK-vector by the presence of 40 micromol/L 5-nitro-2-(3-phenylpropylamino)-benzoic acid but not by 2 mmol/L probenecid. Thus, MRP5 transports the monophosphorylated metabolite of this nucleoside and when MRP5 is overexpressed in colorectal and breast tumors, it may contribute to 5-FU drug resistance.     

The pharmacological profile of (R)-3,4-dihydro-N-isopropyl-3-(N-isopropyl-N-propylamino)-2H-1-benzopyran-5-carboxamide, a selective 5-hydroxytryptamine(1A) receptor agonist.

The pharmacological properties of the 5-hydroxytryptamine (HT)(1A) receptor agonist (R)-3,4-dihydro-N-isopropyl-3-(N-isopropyl-N-propylamino)-2H-1-benzopyran-5-carboxamide (NAE-086) were examined with in vitro and in vivo techniques. Receptor binding studies demonstrated that NAE-086 was a high-affinity and selective 5-HT(1A) receptor ligand with a K(i) value of 4.5 nM in membranes from rat hippocampus. Of 32 other receptors examined NAE-086 had a modest affinity only for the 5-HT(7) receptor (K(i) = 240 nM). NAE-086 inhibited VIP-stimulated adenylyl cyclase activity in GH(4)ZD10 cells with 79% of the efficacy of 5-HT. This inhibition was blocked by the 5-HT(1A) receptor (and beta-adrenoceptor) antagonist (-)alprenolol. A minor metabolite of NAE-086 in rats, (R)-3,4-dihydro-3-(N-isopropyl-N-propylamino)-2H-1-benzopyran-5-carboxamide had a similar receptor profile but had 17 times higher affinity for the 5-HT(1A) receptor (K(i) = 0.26 nM). In vivo, NAE-086 induced all the typical effects of a 5-HT(1A) receptor agonist in rats: it decreased 5-HT synthesis (5-HTP accumulation) and 5-HT turnover (measured as the ratio of 5-hydroxyindoleacetic acid/5-HT), increased corticosterone secretion, induced the 5-HT(1A) syndrome (flat body posture and forepaw treading), inhibited the cage-leaving response, and caused hypothermia. All the responses mediated by postsynaptic 5-HT(1A) receptors were attenuated after single or repeated treatment of the rats with NAE-086. Simultaneously with the development of the tolerance to 5-HT(1A) receptor-mediated responses, 5-HT(2A) receptor-mediated responses were enhanced, as judged from the increased number of spontaneous and/or agonist [1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane]-induced wet-dog shake responses. The significance of this behavioral effect in relation to clinical observations is discussed.     

急性白血病患者多药耐药相关蛋白基因表达及临床意义

目的：研究急性白血病（ＡＬ）多药耐药相关蛋白（ＭＲＰ）基因表达与临床耐药的关系。方法：应用半定量逆转录－聚合酶链反应，检测了６５例ＡＬ患者骨髓和１５名正常人外周血单个核细胞中ＭＲＰ基因的表达。以ＭＲＰ／β２微球蛋白（β２Ｍ）评价ＭＲＰ的表达水平，将ＭＲＰ／β２Ｍ≥０．３表示为ＭＲＰ阳性。结果：复发难治组ＭＲＰ的平均表达水平及阳性率最高，与正常对照组、初治组、长期生存组的ＭＲＰ平均表达水平及阳性率相比均有显著差异（Ｐ＜０．０５），而长期生存组与正常对照组之间无统计学差异。初治组ＭＲＰ阳性病例与ＭＲＰ阴性病例的首次完全缓解率（分别为２５％和８４％）之间有显著差异。ＭＲＰ表达水平在ＦＡＢ各亚型间略有差异。同时检测了６５例ＡＬ患者ｍｄｒ－１基因的表达，发现临床耐药组ＭＲＰ和ｍｄｒ－１的平均表达水平及阳性率均明显高于临床非耐药组，ＭＲＰ和ｍｄｒ－１基因的表达水平之间无相关性（ｒｓ＝０．１６８３）。结论：ＭＲＰ基因过度表达可导致临床耐药，是预后的重要不利因素。     

p16蛋白、雌、孕激素受体在乳癌中的表达

目的 探讨p16蛋白在乳腺癌中的表达及其与ER、PR表达的关系。方法 采用免疫组织法检测84例乳腺癌的p16蛋白、ER和PR。结果 p16阳性率（47．1％）与ER阳性率（39．28％）和PR阳性率（41．7％）相类似。p16阳性病例中巴结转移率（27．5％）远低于p16阴性者（61．36％）,两者差异显著（P＜0．05）。ER与淋巴结转移的关系相似,ER阳性病例的转移率为30．30％,阴性病例转移率为54．96％,两者差异显著（P＜0．05）。结论 p16、ER和PR的表达具有较强的一致性,从p16的异常表达可推测p16基因的突变和缺失可能与乳腺癌的发生、发展有关。p16与ER的表达与淋巴结转移有明显的相关性,p16和ER表达阳性的乳腺癌转移率较低,p16和ER均阴性者淋巴结转移率较高。因此,p16和ER均可作为乳癌预后的指标。     

乳腺癌组织中C-erbB-2,p53,ER和PR的表达及临床意义

目的:探讨C-erb-2,p53,雌激素受体(estrogen receptor,ER)、孕激素受体(progesterone receptor,PR)在乳腺癌中的表达及临床价值.方法:采用免疫组织化学SP法检测246例乳腺癌组织及其癌旁正常乳腺组织中C-erbB-2,p53,ER和PR蛋白的表达情况.结果:在246例乳腺癌中,C-erbB-2,p53,ER和PR的阳性表达率分别为77.6%,73.6%,63.0%和57.7%;C-erbB2和p53阳性表达率在不同临床分期、有无淋巴结转移上差异有统计学意义(P＜0.05);C-erbB-2,p53,ER和PR阳性表达率在不同肿瘤大小上差异无统计学意义(P＞0.05).结论:联合检测乳腺癌C-erbB-2,p53,ER和PR对判断预后和治疗有指导价值.     

Quantitation of doxorubicin uptake, efflux, and modulation of multidrug resistance (MDR) in MDR human cancer cells

P-glycoprotein (Pgp), a membrane transporter encoded by the MDR1 gene in human cells, mediates drug efflux from cells, and it plays a major role in causing multidrug resistance (MDR). Confocal microscopy was used to study in vitro and in vivo drug accumulation, net uptake and efflux, and MDR modulation by P-glycoprotein inhibitors in MDR1-transduced human MDA-MB-435mdr (MDR) cancer cells. The MDR cells were approximately 9-fold more resistant to the anticancer drug doxorubicin than their parental wild-type MDA-MB-435wt (WT) cells. Doxorubicin accumulation in the MDR cells was only 19% of that in the WT cells. The net uptake of doxorubicin in the nuclei of the MDR cells was 2-fold lower than that in the nuclei of the WT cells. Pgp inhibitors verapamil, cyclosporine A, or PSC833 increased doxorubicin accumulation in the MDR cells up to 79%, and it reversed drug resistance in these cells. In living animals, doxorubicin accumulation in MDA-MB-435mdr xenograft tumors was 68% of that in the wild-type tumors. Administration of verapamil, cyclosporine A, or PSC833 before doxorubicin treatment of the animals increased doxorubicin accumulation in the MDR tumors up to 94%. These studies have added direct in vitro and in vivo information on the capacity of the transporter protein Pgp to efflux doxorubicin and on the reversal of MDR by Pgp inhibitors in resistant cancer cells.     

Coexpression of P2X(3) and P2X(2) receptor subunits in varying amounts generates heterogeneous populations of P2X receptors that evoke a spectrum of agonist responses comparable to that seen in sensory neurons

Using voltage-clamp procedures on Xenopus oocytes, agonist-evoked ionic currents by P2X receptors resulting from the coexpression of P2X(2) and P2X(3) subunits were compared against the agonist responses of homomeric P2X(2) and P2X(3) receptors. With the quantity of P2X(3) mRNA kept constant and quantity of P2X(2) mRNA progressively increased, expressed P2X receptors changed from a P2X(3)-like receptor to a P2X(2)-like receptor. In all cases, however, agonist-evoked responses comprised biphasic (fast and slow) currents-the former showing the properties of P2X(3) receptors and latter consistent with the presence of P2X(2) and P2X(2/3) receptors. Using desensitization procedures, the P2X(3)-like fast current was selectively removed to allow the slow current to be studied in isolation. P2X(2/3) receptors were then characterized by slowly inactivating inward currents that were reproducible within 30 s of washout and whose pharmacological profile [selective agonists, Ap(5)A > alpha,beta-methylene ATP > beta,gamma-methylene ATP > UTP; antagonists, TNP-ATP > suramin > or = Reactive blue-2 (RB-2)] contrasted with the profile of P2X(2) receptors (Ap(5)A, alpha,beta-methylene ATP, beta,gamma-methylene ATP, and UTP inactive; antagonists, RB-2 > TNP-ATP > suramin). Thus, our experiments reveal that coexpression of two P2X subunits, which of themselves can generate functional homomeric receptors, results in a complex population of heterogeneous P2X receptors-in this case P2X(2), P2X(3), and P2X(2/3) receptors. Depending on the relative levels of P2X subunit coexpression, the operational profile of the resultant P2X receptors can change from one phenotype to another. This spectrum may explain the variability of agonist responses in small sensory neurons that also express P2X(2) and P2X(3) subunits in different amounts.     

The antinociceptive effect of 2-chloro-2'-C-methyl-N6-cyclopentyladenosine (2'-Me-CCPA), a highly selective adenosine A1 receptor agonist, in the rat

This study was undertaken in order to investigate the effect of 2-chloro-2'-C-methyl-N(6)-cyclopentyladenosine (2'-Me-CCPA), a potent and highly selective adenosine A(1) receptor agonist, on nociceptive responses and on the ongoing or tail flick-related changes of rostral ventromedial medulla (RVM) ON- and OFF-cell activities. Systemic administrations of 2'-Me-CCPA (2.5-5 mg/kg, i.p.) reduced the nociceptive response in the plantar and formalin tests, in a way prevented by DPCPX (3 mg/kg, i.p.), a selective A(1) receptor antagonist. Similarly, intra-periaqueductal grey (PAG) 2'-Me-CCPA (0.5-1-2 nmol/rat) reduced pain behaviour in the plantar and formalin tests, in a way inhibited by DPCPX (0.5 nmol/rat). Moreover, when administered systemically (2.5-5 mg/kg, i.p.) or intra-PAG (0.5-1 nmol/rat) 2'-Me-CCPA increased the tail flick latencies, delayed the tail flick-related onset of the ON-cell burst and decreased the duration of the OFF-cell pause in a dose dependent manner. Furthermore, it decreased RVM ON-cell and increased OFF-cell ongoing activities. The in vivo electrophysiological effects were all prevented by DPCPX (0.5 nmol/rat). This study confirms the role of adenosine A(1) receptors in modulating pain and suggests a critical involvement of these receptors within PAG-RVM descending pathway for the processing of pain.