Shaoyou Chu

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.

Dynamic regulation of gastric surface pH by luminal pH

In vivo confocal imaging of the mucosal surface of rat stomach was used to measure pH noninvasively under the mucus gel layer while simultaneously imaging mucus gel thickness and tissue architecture. When tissue was superfused at pH 3, the 25 microm adjacent to the epithelial surface was relatively alkaline (pH 4.1 +/- 0.1), and surface alkalinity was enhanced by topical dimethyl prostaglandin E2 (pH 4.8 +/- 0.2). Luminal pH was changed from pH 3 to pH 5 to mimic the fasted-to-fed transition in intragastric pH in rats. Under pH 5 superfusion, surface pH was relatively acidic (pH 4.2 +/- 0.2). This surface acidity was enhanced by pentagastrin (pH 3.5 +/- 0.2) and eliminated by omeprazole, implicating parietal cell H,K-ATPase as the dominant regulator of surface pH under pH 5 superfusion. With either pH 5 or pH 3 superfusion (a) gastric pit lumens had the most divergent pH from luminal superfusates; (b) qualitatively similar results were observed with and without superfusion flow; (c) local mucus gel thickness was a poor predictor of surface pH values; and (d) no channels carrying primary gastric gland fluid through the mucus were observed. The model of gastric defense that includes an alkaline mucus gel and viscous fingering of secreted acid through the mucus may be appropriate at the intragastric pH of the fasted, but not fed, animal.

Direct measurement of acid permeation into rat oesophagus

Background and aims: The early responses of the oesophageal mucosa to acid perfusion may predict subsequent pathology. Mucosal responses to luminal acid may result either from acid permeating through the mucosa or from other unknown transduction mechanisms. In order to better understand the dynamics of acid permeation into the oesophageal mucosa, we measured interstitial pH (pHint) of the oesophageal basal epithelial layer, pre-epithelial layer thickness, and blood flow in rats in vivo during luminal acid challenge. A novel confocal microscopic technique was used in vitro to measure pHint from defined cellular sites in response to luminal and basolateral acidification. Methods: 5-(and-6)-Carboxyfluorescein (CF) and carboxy-seminapthorhodofluor-1 (SNARF-1) fluorescence was used to measure pHint by conventional and confocal microscopy, respectively, in urethane anaesthetised rats. Pre-epithelial layer thickness was measured optically with carbon particles as markers. Blood flow was measured with laser Doppler flowmetry. Results: Luminal acidification failed to alter pHint in vivo and in vitro, but pHint was lowered by modest serosal acidification. Pre-epithelial layer thickness and blood flow increased significantly during luminal surface acid perfusion. Indomethacin had no effect on any acid related response. Conclusion: In this first dynamic measurement of oesophageal acid permeation and pre-epithelial layer thickness, pHint was preserved in spite of high luminal acidity by two complementary techniques. Despite the apparent permeability barrier to acid permeation, oesophageal blood flow and thickness responded to luminal acid perfusion.

Dynamic Assessment of Mitoxantrone Resistance and Modulation of Multidrug Resistance by Valspodar (PSC833) in Multidrug Resistance Human Cancer Cells

P-glycoprotein (Pgp), a member of the ATP-binding cassette transporter family, is one of the major causes for multidrug resistance (MDR). We report using confocal microscopy to study the roles of Pgp in mediating the efflux of the anticancer agent mitoxantrone and the reversal of MDR by the specific Pgp inhibitor valspodar (PSC833). The net uptake and efflux of mitoxantrone and the effect of PSC833 were quantified and compared in Pgp-expressing human cancer MDA-MB-435 (MDR) cells and in parental wild-type cells. The MDR cells, transduced with the human Pgp-encoding gene MDR1 construct, were approximately 8-fold more resistant to mitoxantrone than the wild-type cells. Mitoxantrone accumulation in the MDR cells was 3-fold lower than that in the wild-type cells. The net uptake of mitoxantrone in the nuclei and cytoplasm of MDR cells was only 58 and 67% of that in the same intracellular compartment of the wild-type cells. Pretreatment with PSC833 increased the accumulation of mitoxantrone in the MDR cells to 85% of that in the wild-type cells. In living animals, the accumulation of mitoxantrone in MDA-MB-435mdr xenograft tumors was 61% of that in the wild-type tumors. Administration of PSC833 to animals before mitoxantrone treatment increased the accumulation of mitoxantrone in the MDR tumors to 94% of that in the wild-type tumors. These studies have added direct in vitro and in vivo visual information on how Pgp processes anticancer compounds and how Pgp inhibitors modulate MDR in resistant cancer cells.

Endogenous cyclo-oxygenase activity regulates mouse gastric surface pH

In the stomach, production of prostaglandins by cyclo‐oxygenase (COX) is believed to be important in mucosal defence. We tested the hypothesis that endogenous COX activity is required for protective gastric surface pH control. Intact stomachs of anaesthetized mice were perfused with a weakly buffered solution (150 mmNaCl+ 4 mm Homopipes) at pH values from 2.5 to 7.0. Gastric effluents were collected to measure pH and estimate amounts of acid or alkali secretion in nanomoles secreted per minute. A switch from net acid to net alkali secretion was seen in response to acidifying luminal pH with an apparent ‘set point’ between pH 4 and 5. At luminal pH 3, the net alkali secretion (12.7 ± 2.8 nmol OH− equivalents min−1) was abolished (2.2 ± 1.7 nmol OH− min−1) by the non‐specific COX inhibitor indomethacin (5 mg kg−1 I.P.). Similar inhibition was observed using a COX‐1 inhibitor (SC‐560; 10 mg kg−1 I.P.), but not a COX‐2 inhibitor (NS‐398; 10 mg kg−1 I.P.). Subsequent treatment with 16,16‐dimethyl prostaglandin E2 (dm‐PGE2; 1 mg kg−1 I.P.) rescued the alkali secretion (21.8 ± 2.7 nmol OH− min−1). In either the absence or presence of the H+,K+‐ATPase inhibitor omeprazole (60 mg kg−1 I.P.), indomethacin blocked similar amounts of net alkali secretion (10.5 ± 2.7 and 16.4 ± 3.4 nmol OH− min−1, respectively). We also used in vivo confocal microscopy to examine pH near the mucosal surface. The gastric mucosal surface of anaesthetized mice was exposed and mucosal surface pH was imaged using the fluorescence intensity ratio of Cl‐NERF as a pH indicator. Results showed a switch from a continuous net acid to net alkali secretion by the stomach in response to changing superfusate pH from 5 to 3. At luminal pH 3, the relatively alkaline surface pH (4.3 ± 0.1) was acidified (3.6 ± 0.2) by indomethacin, and subsequent dm‐PGE2 restored surface pH (4.2 ± 0.2). We conclude that the pre‐epithelial alkaline layer is regulated by endogenous COX activity.

Transepithelial SCFA Fluxes Link Intracellular and Extracellular pH Regulation of Mouse Colonocytes

We have studied pH regulation in both intracellular and extracellular compartments of mouse colonic crypts, using distal colonic mucosa with intact epithelial architecture. In this work, we question how transepithelial SCFA gradients affect intracellular pH (pHi) and examine interactions between extracellular pH (pHo) and pHi regulation in crypts of distal colonic epithelium from mouse. We studied pH regulation in three adjacent compartments of distal colonic epithelium (crypt lumen, crypt epithelial cell cytosol, and lamina propria) with SNARF-1 (a pH sensitive fluorescent dye), digital imaging microscopy (for pHi), and confocal microscopy (for pHo). Combining results from the three compartments allows us to find how pHi and pHo are regulated and related under the influence of physiological transepithelial SCFA gradients, and develop a better understanding of pH regulation mechanisms in colonic crypts. Results suggest a complex interdependency between SCFA fluxes and pHo values, which can directly affect how strongly SCFAs acidify colonocytes.

TRANSEPITHELIAL SCFA GRADIENTS REGULATE POLARIZED NA/H EXCHANGERS AND PH MICRODOMAINS IN COLONIC EPITHELIA

Short chain fatty acids (SCFAs) stimulate electroneutral sodium absorption by activation of apical Na/H exchange in colonocytes. It is often assumed that activation of Na/H exchange is via an intracellular acidification caused by SCFA uptake. These lecture notes review shortcomings in this model of SCFA-stimulated sodium absorption, revealed by recent reports in the literature. This is supplemented by information generated in our laboratory using both a tissue culture model of colonocytes (HT29-C1 cells) and a native tissue preparation (mouse distal colonic mucosa). In both preparations, evidence suggests that physiologic SCFA gradients may generate pH heterogeneity in aqueous microdomains near the plasma membrane of colonocytes. Finally, direct observation of such extracellular microdomains with confocal microscopy is used to support a new model, in which pH microdomains play an important role in regulating both SCFA fluxes and sodium absorption.

Generation and activity of a humanized monoclonal antibody that selectively neutralizes the epidermal growth factor receptor ligands transforming growth factor-α and epiregulin.

At least seven distinct epidermal growth factor (EGF) ligands bind to and activate the EGF receptor (EGFR). This activation plays an important role in the embryo and in the maintenance of adult tissues. Importantly, pharmacologic EGFR inhibition also plays a critical role in the pathophysiology of diverse disease states, especially cancer. The roles of specific EGFR ligands are poorly defined in these disease states. Accumulating evidence suggests a role for transforming growth factor α (TGFα) in skin, lung, and kidney disease. To explore the role of Tgfa, we generated a monoclonal antibody (mAb41) that binds to and neutralizes human Tgfa with high affinity (KD = 36.5 pM). The antibody also binds human epiregulin (Ereg) (KD = 346.6 pM) and inhibits ligand induced myofibroblast cell proliferation (IC50 values of 0.52 and 1.12 nM for human Tgfa and Ereg, respectively). In vivo, a single administration of the antibody to pregnant mice (30 mg/kg s.c. at day 14 after plug) or weekly administration to neonate mice (20 mg/kg s.c. for 4 weeks) phenocopy Tgfa knockout mice with curly whiskers, stunted growth, and expansion of the hypertrophic zone of growth plate cartilage. Humanization of this monoclonal antibody to a human IgG4 antibody (LY3016859) enables clinical development. Importantly, administration of the humanized antibody to cynomolgus monkeys is absent of the skin toxicity observed with current EGFR inhibitors used clinically and no other pathologies were noted, indicating that neutralization of Tgfa could provide a relatively safe profile as it advances in clinical development.

The glow of the colonic pH microclimate kindled by short‐ chain fatty acids, chloride and bicarbonate

Epithelial cells are interface engines. They sit at the boundary between two distinct compartments (e.g. the intestinal lumen and the body) and consume cellular energy to shuttle solutes and water between the two compartments. Theoretical and experimental evidence suggests that one consequence of this transepithelial transport is a microscopic environment directly adjacent to epithelial cells. This microenvironment can maintain a composition distinct from the bulk solutions because of (1) limited mixing near the cellular surface, (2) fixed surface charges on the membrane, and (3) robust transport across the cell membrane. The concept is important because this is the environment which controls activation of membrane transport to drive transepithelial transport and the uptake of many drugs. Such microenvironments have been proposed to have physiological roles in the stomach, airways and intestines. In this issue of The Journal of Physiology Genz, Engelhardt & Busche explore the extracellular pH directly adjacent to the apical membrane of colonic epithelia (referred to as the pH microclimate). The investigators are not alone in this goal. Early studies with pH-sensitive microelectrodes discovered a relatively constant pH microclimate near the surface epithelium of mammalian colon (McNeil et al. 1987), but results were plagued with questions about the precise location of the electrodes and the spatial resolution of such methods. More recent studies with water-soluble pH-sensitive fluorescent dyes and new modes of microscopy (confocal and digital deconvolution) have successfully measured the dynamic changes of extracellular pH in mouse colonic crypts (Chu & Montrose, 1996) with submicrometre resolution. However, these measurements from the aqueous phase leave lingering doubts about the events directly at the membrane surface. Enter the innovative contribution by Genz et al. who used a near-membrane pH probe composed of a pH-sensitive fluorescent dye coupled to an aliphatic (lipophilic) chain. This idea builds on a landmark paper by Dragsten et al. (1981) which shows that long saturated fatty acids stay in the outer leaflet of the membrane bilayer and hence can be restricted to either the apical or basolateral domain in epithelial cells. Genz et al. extended this observation to develop a method for measurement of pH with 5-N-hexadecanoyl-aminofluorescein (HAF), which inserts into the outer leaflet of the apical membrane of isolated guinea-pig colonic epithelium. The authors carefully documented the site of dye insertion, tissue viability, and the quality of fluorescence response to changes in pH in the range of pH 6-8. Thus, the authors established a method for the non-invasive measurement of pH directly at the outer surface of the apical membrane of the surface epithelium. The authors applied the method to investigate the effects of chloride, bicarbonate and butyrate on the pH microclimate. An important contribution in the paper is a novel observation regarding chloride. Genz et al. show for the first time that a Cl−-HCO3− exchanger is probably a major contributor to surface pH microclimate. This is an important observation regarding the transporter widely believed to mediate colonic bicarbonate secretion. In contrast, there was little support for expectations that H+-K+-ATPase was the dominant apical transporter mediating proton efflux (and thereby supplying luminal protons to sustain SCFA absorption) in guinea-pig distal colon (Englelhardt et al. 1993). Genz et al. report that H+-K+-ATPase had only a slight influence on the surface pH. The colonic lumen is a bacterial broth that metabolizes undigested protein and carbohydrate to produce short-chain fatty acids (SCFAs) such as acetate, propionate and butyrate. SCFAs hold fascination for investigators as the most abundant anions and osmolytes in the colonic lumen, as physiological stimulators of sodium absorption and bicarbonate secretion, and because they hold a tantalizing link to the health of the colon which goes awry in ulcerative colitis. Genz et al. found that a SCFA (butyrate) qualitatively affected surface pH in a similar way to HCO3, which gives important corroboration for earlier observations by Chu & Montrose (1996) with a different method, a different species (mouse), and a different part of the epithelium (surface versus crypt). Results of Genz et al. hint that Cl−-SCFA exchange may explain part of the effects of luminal chloride on the pH microclimate, although evidence is also presented that other (Cl− independent) routes of SCFA flux must also be important to microclimate regulation. However, it is exciting to have a putative physiological role for the Cl−-SCFA exchange reported in colonic membrane vesicles by Rajendran & Binder (1994). The contribution of Genz et al. strengthens the belief that regulated extracellular microenvironments are part of the cellular milieu, at least for transporting epithelia. The existence of a microscopic space with its own behaviour is undeniably awkward for studies of cell and tissue response. However, it should be recognized that microenvironments also provide a new opportunity. Disturbances in microenvironment may contribute to disease states by causing changes in drug uptake, cell/tissue viability, or changing the activity of membrane transporters. Optical approaches have become the preferred tools for non-invasively measuring microenvironments, and this fuels their ascent as powerful and essential tools for the study of ion transport by cells and tissues. We now have the opportunity for a clearer vision of the world from a cellular perspective.

Correlation of surface pH versus mucus gel thickness by in vivo confocal microscopy of rat gastric mucosa

and HM-CAP, but the FLEX PACK assay failed to detect two (33%). Conclusions: (1) The Chiron lysate and HM-CAP are equally sensitive in detecting HP antibody but the lysate assay is twice as specific; (2) the FLEX PACK assay is significantly less sensitive than the other two assays; (3) 29% of pediatric patients with abdominal pain have antibodies to CagA or VacA. These patients are accurately detected by Chiron lysate or HM-CAP assays, but not by FLEX PACK. This study was funded in part by a grant from EPI.