Multidrug resistance gene and P-glycoprotein expression in gastric adenocarcinoma and precursor lesions

Overexpression of the Multiple Drug Resistance gene (MDR1) has been proposed as a major mechanism related to both intrinsic and acquired resistance to chemotherapeutic agents. The gene product is a membrane protein (P-glycoprotein), that acts as an energydependent drug efflux pump decreasing drug accumulation in resistant tumor cells. We have characterized MDR1 and P-Glycoprotein expression in human gastric adenocarcinoma and in precursor lesions. MDR1 mRNAs, analyzed by dot-blot technique, were detected in 9 of 10 non-tumoral gastric mucosae and in 8 of 10 gastric adenocarcinomas. Immunohistochemical analysis, using the MRK16 monoclonal antibody, revealed heterogeneous expression of P-Glycoprotein in individual cells. The P-Glycoprotein was found on the surface of cells of gastric areas with intestinal metaplasia subtype III. This type of intestinal metaplasia, also called “colonic metaplasia”, has been strongly associated with a high risk for the development of gastric cancer. The fact that the P-Glycoprotein was detected in this precursor lesion is consistent with the intestinal metaplasia dysplasia and carcinoma sequence proposed in the histogenesis of this tumor. The finding that P-Glycoprotein was heterogeneously expressed in malignant cells of some gastric adenocarcinomas also suggests that this transporter system probably contributes to primary and secondary multidrug resistance in this neoplasm.     

Multidrug resistance P-glycoprotein is not involved in cholesterol esterification

The aim of the present paper is to reinvestigate the role of multidrug resistance P-glycoprotein MDR1 and MDR-associated protein (MRP1) in cholesterol esterification using well-characterized inhibitors. Using specific substrate efflux assay, we show that GF120918 (0.2 microM) and probenecid (5 mM) were specific inhibitors of MDR1 and MRP1, respectively. In HepG2 cells, neither of them affect the esterification of cholesterol derived from the uptake of cholesterol-rich lipoprotein, while both verapamil (100 microM) and progesterone (100 microM) were able to inhibit cholesterol esterification. Similar results were obtained with verapamil, progesterone, and GF120918 in the MDR1-overexpressing cells MCF7/ADR. The capacity of progesterone to reduce cholesterol esterification is not correlated with its ability to inhibit MDR1 but is rather due to direct inhibition of acyl-CoA:cholesterol acyltransferase (ACAT). We conclude that the esterification of cholesterol is not correlated with MDR1 or MRP1 activity, thus excluding their role in the intracellular transport of endocytosis-derived cholesterol.     

Developmental expression of P-glycoprotein (multidrug resistance gene product) in the rat brain.

P-Glycoprotein (PGP), a product of the multidrug resistance gene (mdr), acts as an adenosine triphosphate-dependent drug efflux system in cells. Initially, PGP was found in cancer cells, but it is now known that PGP is richly distributed in the adult brain. Passage to the central nervous system is limited by the blood-brain barrier (BBB), and mdr1 gene-deficient mice showed up-regulation of BBB permeability. In this study, we examined the expression and localization of PGP in the rat brain during development. PGP protein was predominantly detected in the membrane fraction of the adult rat brain, although it was also faintly detected in the cytosolic fraction. PGP protein in the membrane fraction was undetectable in the embryo and early stages of postnatal development by immunoblotting studies, was first detected on postnatal day (P) 7, and then gradually increased to reach a plateau. Such changes were observed commonly in the cerebral cortex, hippocampus, and cerebellum. Immunohistochemical studies showed that PGP immunoreactivity was first detected on P7, and intense PGP immunoreactivity was observed in the adult rat brain. Double-immunolabeling studies revealed that PGP was colocalized with von Willebrand factor-immunoreactive capillaries. We further examined the colocalization of PGP and astrocytes using glial fibrillary acidic protein (GFAP) as a marker. Three-dimensional analysis showed that the GFAP-immunoreactive astrocytes possessed fine processes which ensheathed capillaries, but the PGP immunoreactivity did not colocalize with the GFAP immunoreactivity. These results indicate that PGP expression increased with postnatal development and is localized in the brain capillaries.     

Multidrug resistance P-glycoprotein inhibits the antiapoptotic action of mitochondria-targeted antioxidant SkQR1

Mitochondria-targeted antioxidants of the SkQR1 family, being accumulated in energized mitochondria, protect cells from oxidative stress by increasing the level of reduced glutathione and decreasing the cell-damaging effect induced by hydrogen peroxide. Using various human transformed cell lines and SkQR1 (a fluorescent member of the SkQ family), we show that SkQRI is ejected from chemotherapy-resistant cells by P-glycoprotein - one of the main transport proteins determining multidrug resistance typical for many neoplastic cells. It is also shown that SkQR1 ejection is neutralized by P-glycoprotein inhibitors (verapamil and pluronic L61). In experiments on K562 cells, it was found that the subline sensitive to chemotherapy is protected by SkQRI from apoptotic action of hydrogen peroxide. Protection of the resistant subline occurs only after inhibition of P-glycoprotein.     

Multidrug resistance (MDR1) P-glycoprotein enhances esterification of plasma membrane cholesterol

Class I P-glycoproteins (Pgp) confer multidrug resistance in tumors, but the physiologic function of Pgp in normal tissues remains uncertain. In cells derived from tissues that normally express Pgp, recent data suggest a possible role for Pgp in cholesterol trafficking from the plasma membrane to the endoplasmic reticulum. We investigated the esterification of plasma membrane cholesterol under basal conditions and in response to sphingomyelinase treatment in transfected and drug-selected cell lines expressing differing amounts of functional class I Pgp. Compared with parental NIH 3T3 fibroblasts, cells transfected with human multidrug resistance (MDR1) Pgp esterified more cholesterol both without and with sphingomyelinase. Esterification also was greater in drug-selected Dox 6 myeloma cells than parental 8226 cells, which express low and non-immunodetectable amounts of Pgp, respectively. However, no differences in total plasma membrane cholesterol were detected. Transfection of fibroblasts with the multidrug resistance-associated protein (MRP) did not alter esterification, showing that cholesterol trafficking was not generally affected by ATP-binding cassette transporters. Steroidal (progesterone, dehydroepiandrosterone) and non-steroidal antagonists (verapamil, PSC 833, LY335979, and GF120918) were evaluated for effects on both cholesterol trafficking and the net content of 99mTc-Sestamibi, a reporter of drug transport activity mediated by Pgp. In Pgp-expressing cells treated with nonselective and selective inhibitors, both the kinetics and efficacy of inhibition of cholesterol esterification differed from the antagonism of drug transport mediated by Pgp. Thus, although the data show that greater expression of class I Pgp within a given cell type is associated with enhanced esterification of plasma membrane cholesterol in support of a physiologic function for Pgp in facilitating cholesterol trafficking, the molecular mechanism is dissociated from the conventional drug transport activity of Pgp.     

Smad4 expression in gastric adenoma and adenocarcinoma: frequent loss of expression in diffuse type of gastric adenocarcinoma

Smads are signal transducers for the members of the TGF-beta superfamily. Of these Smads, Smad4 is essential for TGF-beta signaling. The purpose of this study was to elucidate Smad4 expression and localization in 65 gastric adenomas, 49 intestinal-type and 39 diffuse type of gastric adenocarcinomas (including 12 cases of fresh frozen tissue) using Real-time RT-PCR and immunohistochemistry. Real-time RT-PCR showed that intestinal type gastric adenocarcinomas have higher Smad4 mRNA expression than diffuse type gastric adenocarcinomas. Immunohistochemical stain for Smad4 revealed that expression of Smad4 was significantly lower in diffuse-type gastric adenocarcinoma than intestinal-type gastric adenocarcinomas. Also, higher Smad4 protein expression in intestinal type gastric adenocarcinomas than overall gastric adenoma was noted. The rate of reduced Smad4 expression was higher in advanced gastric cancer than early gastric cancer. These results suggest that Smad4 might play different roles in human gastric carcinogenesis, especially between intestinal type and diffuse type of gastric adenocarcinoma.     

Multidrug resistance gene expression is controlled by steroid hormones in the secretory epithelium of the uterus

The multidrug resistance (mdr) gene family has been shown to encode a membrane glycoprotein, termed the P-glycoprotein, which functions as a drug efflux pump with broad substrate specificity. This multigene family is expressed in a tissue-specific fashion in a wide variety of normal and neoplastic tissues. The regulation of mdr gene expression in normal tissues is not understood. We have recently shown that mdr mRNA and the P-glycoprotein increases dramatically in the secretory luminal and glandular epithelium of the gravid murine uterus. This observation has suggested that mdr gene expression in the uterus is controlled by the physiologic changes associated with pregnancy. This report now demonstrates that mdr mRNA and P-glycoprotein are induced at high levels in the uterine secretory epithelium by the combination of estrogen and progesterone, the major steroid hormones of pregnancy. This regulation of mdr gene expression in the uterus does not require any other contribution from the fetus or placenta. The data indicate that this gene locus is hormonally responsive to estrogen and progesterone in the uterine secretory epithelium, suggesting an important and physiologically regulated role during pregnancy.     

Multidrug resistance in fungi

Antibiotic and synthetic chemotherapeutic resistance in pathogenic yeast becomes one of the biggest challenges for the modern chemotherapy. An increasing number of pathogenic yeast and filamentous fungi resistant to the action of the majority of currently used drugs is isolated in clinics nowadays. Among variety of the resistance mechanisms, the most dangerous grows to be the multidrug resistance. The most important mechanism of the multidrug resistance is the overexpression of membrane proteins participating in the active efflux of drugs out of the cells subjected to chemotherapy. Representatives of two classes of multidrug efflux transporters, ABC and MFS, have been identified in fungi. One of the most important strategies for overcome the phenomenon of multidrug resistance in pathogenic fungi, is the use of chemical compounds co-administrated with chemotherapeutics which are able to restore drug susceptibility in multidrug resistant cells. Mode of action of these chemical compounds may be very diverse, from the substrate competition, through the influence on the membrane fluidity, to the multidrug transporters activity modulation. This paper presents a review of the current knowledge on proteins contributing to fungal multidrug resistance and strategies for overcoming multidrug resistance by pharmacological intervention.     

P-糖蛋白对结肠癌多药耐药的影响

结肠癌是常见的消化道恶性肿瘤。对术后患者以及无法采用手术治疗的患者,临床多采用化疗、放疗等综合性治疗方法。随着大量化疗药物在临床的广泛使用,结肠癌多药耐药性成为化疗失败的最主要原因。研究表明,P-糖蛋白(P-glycoprotein, P-gp)作为ATP结合盒(ABC)转运蛋白超家族成员之一,与多种肿瘤的多药耐药相关,其介导的多药耐药已经成为目前研究的热点。本文旨在通过对P-糖蛋白的结构、耐药机制以及逆转P-糖蛋白介导的结肠癌多药耐药新发现进行阐述,引导读者对P-糖蛋白在结肠癌多药耐药中的作用有更深入的了解。     

Tissue-specific expression of the rat secretin precursor gene.

Secretin is a 27-amino acid gastrointestinal hormone that stimulates the secretion of bicarbonate-rich pancreatic fluid. We previously demonstrated that the secretin precursor gene is expressed in the brain as well as in the small intestine. In this study, we demonstrated that the abundance of secretin precursor mRNA in the heart, lung, and kidney was comparable to that of the small intestine. The nucleotide sequences of the coding regions of the secretin precursor mRNAs in these tissues were identical to those of the small intestine, indicating that secretin precursor proteins produced in these tissues are identical to those in the small intestine. This is the first report that the secretin precursor gene is also expressed in the heart, lung, kidney, and testis as well as in the gastrointestinal tract and brain.     

Structure and expression of the human MDR (P-glycoprotein) gene family.

The human MDR (P-glycoprotein) gene family is known to include two members, MDR1 and MDR2. The product of the MDR1 gene, which is responsible for resistance to different cytotoxic drugs (multidrug resistance), appears to serve as an energy-dependent efflux pump for various lipophilic compounds. The function of the MDR2 gene remains unknown. We have examined the structure of the human MDR gene family by Southern hybridization of DNA from different multidrug-resistant cell lines with subfragments of MDR1 cDNA and by cloning and sequencing of genomic fragments. We have found no evidence for any other cross-hybridizing MDR genes. The sequence of two exons of the MDR2 gene was determined from genomic clones. Hybridization with single-exon probes showed that the human MDR1 gene is closely related to two genes in mouse and hamster DNA, whereas MDR2 corresponds to one rodent gene. The human MDR locus was mapped by field-inversion gel electrophoresis, and both MDR genes were found to be linked within 330 kilobases. The expression patterns of the human MDR genes were examined by enzymatic amplification of cDNA. In multidrug-resistant cell lines, increased expression of MDR1 mRNA was paralleled by a smaller increase in the levels of MDR2 mRNA. In normal human tissues, MDR2 was coexpressed with MDR1 in the liver, kidney, adrenal gland, and spleen. MDR1 expression was also detected in colon, lung, stomach, esophagus, muscle, breast, and bladder.     

Multidrug resistance in Gram-negative bacteria

Broadly specific, so-called multidrug, efflux mechanisms are now known to contribute significantly to intrinsic and acquired multidrug resistance in a number of Gram-negative bacteria, and the boom in bacterial genomics has confirmed the distribution of these systems in all bacteria. This broad distribution of multidrug transporters lends a certain credibility to suggestions that they play a housekeeping role in the cell, beyond any contributions they may make to antimicrobial efflux and resistance. In many instances, these transporters are dispensable, arguing against their carrying out essential cellular functions; nevertheless, the multiplicity of these broadly specific export systems within a given microorganism, often with overlapping substrate specificity, may explain the dispensability of individual exporters. Whatever their intended function, however, their conservation in so many organisms highlights their probable general importance in antimicrobial resistance, particularly in Gram-negative bacteria whose outer membranes work synergistically with many of these export systems to promote drug exclusion.     

Multidrug resistance in Entamoeba histolytica

Drug resistance in protozoan parasites has been emerging in the past decade as an obstacle to their control. Amoebiasis, caused by Entamoeba histolytica, is a worldwide disease that provokes high rates of morbidity and mortality. Reports of failed drug treatment and differences in drug susceptibilities among E. histolytica strains probably herald the development of drug resistance in this parasite. In this review, Esther Orozco and co-workers summarize recent progress on the elucidation of physiological and molecular evidence of multidrug resistance in this parasite.     

Enkephalin precursor gene expression in postmeiotic germ cells

Preproenkephalin mRNA was detected in rat testis after postnatal day 30. A high abundance of preproenkephalin mRNA was present in the spermatids (i.e., spermatogenic cells at postmeiotic stage) purified from mature rat testis. The mRNA size of the spermatids was about 1900 bases, which was larger than that of other tissues expressing the preproenkephalin gene. The spermatids contained small amounts of enkephalin-containing proteins, but little or no Met-enkephalin. Preproenkephalin gene expression in the germ cells suggests the connection between gametogenesis and nervous system.     

Variation in expression of gastric leptin according to differentiation and growth pattern in gastric adenocarcinoma

Leptin is an adipocyte-derived hormone that regulates body fat stores and feeding behavior. The presence of leptin in stomach epithelium was recently demonstrated in the rat and humans, and gastric leptin has been linked to the control of meal size, local inflammatory responses, and paracrine and autocrine functions through leptin receptors in the stomach. We compared the expression patterns of leptin and of the long variant of the leptin receptor (Ob-Rb) between areas with non-ulcerated mucosa and with hyperplastic polyps, adenoma, or adenocarcinoma to evaluate the expression relative to different disease states. Leptin and Ob-Rb were expressed in hyperplastic polyps, adenoma, and adenocarcinoma. In the gastric adenocarcinoma, leptin was expressed significantly less in the poorly differentiated and diffuse-type groups than in the well-differentiated and moderately differentiated groups or in the intestinal type. Based upon our findings, we suggest the possibility that leptin expression can have a pathophysiologic role about the differentiation or growth pattern of gastric adenocarcinoma. A further series of experiments is necessary to elucidate the pathophysiological role of leptin in the differentiation of gastric adenocarcinoma.     

Increase of PTEN gene expression in insulin resistance.

Phosphatase and tensin homologue deleted from chromosome ten (PTEN) has recently been characterized as a regulator of insulin sensitivity in the insulin target tissue. However, whether PTEN gene expression is changed in insulin resistance remains unclear. We observed that both the mRNA and protein level of PTEN in soleus muscle isolated from the obese Zucker rats (Fa/Fa) were increased compared to the age-matched lean group. Similarly, both the mRNA and protein level of PTEN in soleus muscle of the fructose-fed lean Zucker rats (Fa/Fa) showing the higher glucose-insulin index were higher than that of the regular chow fed group. These results suggest that increase of PTEN gene expression seems to be related to the development of insulin resistance.