Rat Ascites Hepatoma Cell Line Research Articles

Effect of Apple Polyphenol Extract on Hepatoma Proliferation and Invasion in Culture and on Tumor Growth, Metastasis, and Abnormal Lipoprotein Profiles in Hepatoma-Bearing Rats

The effect of apple polyphenol extract (APE) on the proliferation and invasion of a rat ascites hepatoma cell line of AH109A was examined in vitro. APE suppressed both the hepatoma proliferation and invasion in a dose-dependent manner up to 200 mug/ml. Serum obtained from rats orally given APE also inhibited hepatoma proliferation and invasion when added to the culture medium. Subsequently, the effect of dietary APE on growth and the metastasis of AH109A hepatomas were investigated in vivo. APE reduced the growth and metastasis of solid hepatomas and significantly suppressed the serum lipid peroxide level in rats transplanted with AH109A. APE also suppressed the serum very-low-density lipoprotein + low-density lipoprotein (VLDL + LDL)-cholesterol level. These in vitro and in vivo findings suggest that APE has anti-hepatoma activities.

Effects of a fish oil–based emulsion on rat hepatoma cell invasion in culture

Objective Total parenteral nutrition containing a lipid emulsion is often employed after surgical tumor resection. This study investigated the effects of a fish oil–based infusion on rat hepatoma cell invasion. Methods Rat ascites hepatoma cell line AH109A was precultured with a fish oil–based or safflower oil–based emulsion for 48 h. Changes in membranous fatty acid composition were evaluated by gas chromatography. The invasiveness of hepatoma cells was assessed by coculturing with mesentery-derived mesothelial cells. To examine ex vivo effects of the fish oil–based infusion on hepatoma invasion, sera were prepared from rats infused with fish oil– or safflower oil–based emulsion and the effects of these sera were assessed. To clarify the mechanism of inhibition of invasion by the fish oil–based emulsion, the effects of prostaglandin (PG) E 2 and PGE 3 on invasion were examined. Results Pretreatment with the fish oil–based emulsion reduced invasiveness without affecting growth compared with the safflower oil–based emulsion. Pretreatment with the sera from rats infused with the fish oil–based emulsion also reduced invasiveness compared with the sera from rats infused with the safflower oil–based emulsion. The addition of PGE 2 eliminated the inhibitory effect of the fish oil–based emulsion, and the addition of PGE 3 reduced the invasiveness of hepatoma cells pretreated with the safflower oil–based emulsion. Conclusion These results suggest that the fish oil–based emulsion may have anti-invasive effects. Changes in the membranous fatty acid composition and consequent changes in the prostaglandins produced may be involved in this inhibitory effect.

Anti-Invasive Activity of Niacin and Trigonelline against Cancer Cells

The effects of niacin, namely, nicotinic acid and nicotinamide, and trigonelline on the proliferation and invasion of cancer cells were studied using a rat ascites hepatoma cell line of AH109A in culture. Niacin and trigonelline inhibited the invasion of hepatoma cells at concentrations of 2.5-40 microM without affecting proliferation. Hepatoma cells previously cultured with a reactive oxygen species (ROS)-generating system showed increased invasive activity. Niacin and trigonelline suppressed this ROS-potentiated invasive capacity through simultaneous treatment of AH109A cells with the ROS-generating system. The present study indicates for the first time the anti-invasive activities of niacin and trigonelline against cancer cells.

Resveratrol inhibits hepatoma cell invasion by suppressing gene expression of hepatocyte growth factor via its reactive oxygen species-scavenging property

Resveratrol, present in grapes and other plants, is a polyphenolic compound with strong antioxidative activity. In our previous studies, we found that reactive oxygen species (ROS) accelerated the invasive capacity of a rat ascites hepatoma cell line of AH109A in culture and that resveratrol and resveratrol-loaded rat sera suppressed the ROS-potentiated invasion of the hepatoma cells. To study mechanisms by which resveratrol and its in vivo metabolite(s) suppress the invasion, we estimated intracellular peroxide level and expression of hepatocyte growth factor (HGF), a known cell motility factor, in AH109A cells. Exogenously added ROS promoted the intracellular peroxide level and the expression of HGF. Resveratrol and resveratrol-loaded rat sera canceled the rise in the peroxide level and HGF expression in ROS-stimulated tumor cells. These results suggest an involvement of the antioxidative property of resveratrol and sera from rats orally given resveratrol in their suppressive effects on ROS-potentiated invasion of AH109A cells.

Inhibitory effect of coffee on hepatoma proliferation and invasion in culture and on tumor growth, metastasis and abnormal lipoprotein profiles in hepatoma-bearing rats.

We have already reported that instant coffee powder (ICP) and ICP-loaded rat sera could suppress proliferation and invasion of rat ascites hepatoma cell line of AH109A in vitro. In this report, we examined the mechanisms for suppression of tumor cell proliferation and invasion by ICP, and the effect of ICP on in vivo tumor growth, metastasis and abnormal lipoprotein profiles in hepatoma-bearing rats. ICP, when directly added to the culture media, induced cell cycle arrest (elongation of S phase) at a lower concentration (0.3 mg/mL) and apoptosis at a higher concentration (0.6-1.2 mg/mL). ICP and ICP-loaded rat sera showed reactive oxygen species (ROS)-scavenging property and canceled the enhancement of invasive activity of hepatoma cells induced by ROS in vitro. These results suggest that ICP suppresses the proliferation by inducing cell cycle arrest and apoptosis, and the invasion by scavenging ROS and that ICP could retain these properties after their gastrointestinal absorption. The hepatoma-bearing rats were fed with a 20% casein diet (20C) or 20C supplemented with 0.1%, ICP for 14 d. Dietary ICP significantly reduced solid tumor growth and tended to reduce hepatoma metastases to lung and lymphatic nodes, suggesting that ICP could suppress tumor cell proliferation and invasion in vivo. In addition, dietary ICP significantly increased serum high-density lipoprotein (HDL)-cholesterol and tended to reduce very low-density and low-density lipoprotein (VLDL+LDL)-cholesterol, resulting in amelioration of abnormal lipoprotein profiles occurred in hepatoma-bearing rats. In conclusion, ICP has the ability to induce cell cycle arrest and apoptosis in hepatoma cells and to suppress tumor cell invasion by reducing oxidative stresses in vitro, and it could also exhibit these effects in vivo, leading to the inhibition of tumor growth and metastases.

Isolation and Characterization of Subpopulations of Rat Ascites Hepatoma Cell Line of AH109A with Different Metastatic Potentials.

Rat ascites hepatoma cell line of AH109A proved to be divided into two subpopulations with different invasive and metastatic potentials, when cultured in the medium containing allogeneic rat sera. One population adheres to the culture dish, actively extending pseudopodia, and the other remains in a floating state. Utilizing this character, we have separated these two populations. After three successive separation steps, adhesive AH109A cells and floating AH109A cells were obtained. Adhesive AH109A cells proliferated more rapidly and invaded more actively than did floating AH109A cells. Adhesive AH109A cells metastasized mainly to lung, while floating AH109A cells to mesentery, when intravenously injected into tail veins. Histological studies revealed that adhesive AH109A cells showed lymphatic metastases to lung. These results suggest that the two populations separated from parental AH109A cells provide good models for the study of tumor invasion and tissue-specific metastasis and that adhesive AH109A cells can be used for the creation of lymphatic metastasis model of rats.

Potentiation of invasive activity of hepatoma cells by reactive oxygen species is mediated by autocrine/paracrine loop of hepatocyte growth factor

We have already reported that reactive oxygen species (ROS) promote rat ascites hepatoma cell invasion beneath mesentery-derived mesothelial cell monolayer. To investigate the mechanism for this, we examined the involvement of motility factors, particularly hepatocyte growth factor (HGF). Rat ascites hepatoma cell line of AH109A expressed HGF and c-Met mRNAs. Treatment with ROS augmented amounts of HGF mRNA in AH109A and HGF concentration in the medium. ROS also induced HGF gene expression in mesothelial cells. Exogenously added HGF enhanced invasive activity of AH109A cells, but exerted no effect on proliferation. AH109A cells pretreated with ROS showed an increased invasive activity, which was cancelled by simultaneous pretreatment with anti-HGF antibody. These results suggest that the invasive activity of AH109A is mediated by the autocrine and paracrine pathways of HGF, and ROS potentiate invasive activity by inducing gene expression of HGF in AH109A and mesothelial cells.

Induction of Tumor Necrosis Factor Production and Antitumor Effect by Cabbage Extract

The effect of cabbage extract on the production of tumor necrosis factor and its implication in the antitumor effect were examined in vitro and in vivo. Cabbage extract stimulated the production of tumor necrosis factor by rat spleen cells and showed cytotoxic activity in a rat ascites hepatoma cell line (AH109A) when hepatoma cells were cultured with cabbage-stimulated spleen cells. When the extract was administered orally to AH109A-bearing rats in combination with lipopolysaccharide injection, the hepatoma weights were reduced to one-half of the vehicle control. The cytotoxic activity of tumor-infiltrating macrophages was induced by simultaneous treatment with cabbage extract and lipopolysaccharide. These results indicate that cabbage extract contains macrophage-stimulating component(s) and can implement the antitumor effect by stimulating the cytotoxicity of tumor-infiltrating macrophages.

Effects of dietary powdered green tea and theanine on tumor growth and endogenous hyperlipidemia in hepatoma-bearing rats.

The effects of dietary powdered green tea (PGT) and theanine on in vivo hepatoma growth and cancerous hyperlipidemia were investigated in rats that had been implanted with a rat ascites hepatoma cell line of AH109A cells. The hepatoma-bearing rats were fed with a 20% casein diet (20C), 20C containing 2% PGT, or 20C containing 0.1% theanine for 14 days. Dietary PGT significantly and time-dependently reduced the solid tumor volume and weight as did dietary theanine. The hepatoma-induced endogenous hyperlipidemia, which was characterized by rises in the serum cholesterol (hypercholesterolemia) and triglyceride (hypertriglyceridemia) levels, was significantly suppressed by PGT and theanine supplementation. Bile acid excretion into the feces was significantly higher in the PGT- and theanine-fed rats than in the control rats. This inhibition of hypercholesterolemia may have resulted from tumor growth suppression as well as increased excretion of steroids from the body. These results suggest that PGT had both anti-proliferative activity toward hepatoma cells and hypolipidemic activity in the hepatoma bearing rats. They also suggest that theanine was, at least in part, responsible for the PGT actions.

Inhibitory effects of theanine and sera from theanine-fed rats on receptor-mediated cancer cell invasion beneath mesothelial-cell monolayers.

To investigate the bioavailability and mode of action of theanine against cancer, we examined in vitro and ex vivo effects of theanine on invasion of a rat ascites hepatoma cell line of AH109A. Theanine dose-dependently inhibited the invasion of AH109A cells across rat mesentery-derived mesothelial-cell (M-cell) monolayers without restraining AH109A cell proliferation in vitro. Rat sera obtained after oral intubation of theanine also inhibited the invasion. A competitive N-methyl-D-aspartate (NMDA) type glutamate receptor antagonist, (+/-) 2-amino-5-phosphonopentanoic acid (AP-5), dose-dependently counteracted the theanine-mediated in vitro and ex vivo inhibition of AH109A invasion. A competitive non-NMDA type glutamate receptor antagonist, 6,7-dinitroquinoxaline 2,3-dione (DNQX), did not affect this inhibition by theanine in vitro. These results suggest that the inhibition of AH109A invasion by theanine may be mediated by the NMDA receptor of AH109A.

Inhibition of hepatoma cell invasion beneath mesothelial-cell monolayer by sera from tea- and related component-treated rats and their modes of action.

The bioavailability and action of teas on the invasion of a rat ascites hepatoma cell line, AH109A, were determined and their modes of action were by co-culturing the cancer cells with a rat mesentery-derived mesothelial-cell (M-cell) monolayer in the presence of sera from rats orally given teas and their component, (-)-epigallocatechin gallate (EGCG). The rat sera obtained 2 and 5 hr after oral intubation of a low concentration of green, oolong, or black tea, or EGCG significantly inhibited AH109A invasion underneath the M-cell monolayer. These sera showed a time-dependent and significant inhibitory effect on the AH109A invasion. The 2-hr sera and 2.5 muM EDTA in the medium completely eliminated the enhancement of AH109A invasion induced by a reactive oxygen species (ROS)-generating system. These results show that the inhibition of relevant ROS-potentiated invasion of AH109A cells across the M-cell monolayer may be due to the antioxidative action of EGCG, the in vivo metabolites, and tea-induced changes in the endogenous substances. The results suggest that the drinking of tea in daily life may have certain preventive and therapeutic effects against cancer cell invasion.

Suppression of adhesion and invasion of hepatoma cells in culture by tea compounds through antioxidative activity

To determine the actions of tea components on the invasion of a rat ascites hepatoma cell line of AH109A and to understand their modes of action, the cancer cells were co-cultured with a rat mesentery-derived mesothelial cell monolayer in the presence of tea components. The synergistic effects of (−)-epicatechin (EC) with (−)-epigallocatechin gallate (EGCG) on AH109A invasion were demonstrated. Further study showed that 10 μM of EGCG or theaflavins, or 2.5 μM of ethylenediaminetetra-acetic (EDTA) entirely abolished the increase in AH109A adhesion and invasion stimulated by reactive oxygen species (ROS) from the hypoxanthine–xanthine oxidase system. Our results suggest that OH −- and other ROS-scavenging activity of EGCG and theaflavins may be responsible for the inhibition of OH −- and related ROS-potentiated AH109A adhesion and invasion to the cultured rat mesothelial cell monolayer.

Inhibitory effects of chlorogenic acid and its related compounds on the invasion of hepatoma cells in culture.

Actions of chlorogenic acid, a major component of coffee, andits constituents, caffeic and quinic acids, on theproliferation and invasion of AH109A, a rat ascites hepatomacell line, were investigated using in vitro assay systems. Allthree components suppressed the AH109A invasion atconcentrations of 5-40 muM without altering the cellproliferation. At the concentration of 10 muM, chlorogenic,caffeic and quinic acids significantly (P < 0.05) suppressedthe invasion by 68%, 36% and 31%, respectively, implying thatthe suppressive effect of chlorogenic acid on the AH109Ainvasion might result from the additive effects of itsconstituents, caffeic and quinic acids. At the concentrationof 10 muM, cinnamic acid and p-coumaric acid (4-hydroxycinnamicacid) exerted no or little influence on the invasion, whereascaffeic acid (3,4-dihydroxycinnamic acid) significantly (P <0.05) suppressed it, suggesting the possible involvement ofthe 3,4-dihydroxy group of caffeic acid in the suppression.Chlorogenic acid was thus demonstrated to be one of thechemical entities in coffee suppressing the hepatoma invasionin vitro, and both of its constituents, caffeic and quinicacids, to be responsible for the anti-invasive activity. Theseresults suggest the existence of nutritionally andpharmacologically important substances in coffee which controltumor cell invasion.

Effects of green, oolong and black teas and related components on the proliferation and invasion of hepatoma cells in culture.

The effects of teas and related components on the proliferation and invasion of cancer cells were examined by employing both in vitro proliferation and invasion assay systems. Powdered green, oolong and black tea extracts dose-dependently inhibited proliferation and invasion of a rat ascites hepatoma cell line of AH109A but did not affect the proliferation of the normal mesentery-derived mesothelial cells (M-cells) isolated from rats; higher concentrations of powdered oolong and black teas could restrain the proliferation of another tumor cell line of L929. The AH109A cells were found to penetrate underneath the monolayer of M-cells in the presence of 10% calf serum. When each rat serum obtained at 0.5, 1, 2, 3 and 5 h after oral intubation of each tea extract was added to the culture media instead of calf serum at a concentration of 10%, both the invasion and proliferation of AH109A were significantly suppressed. These ex vivo results suggest the potential bioavailability of effective tea components in rats. Furthermore, (-)-epigallocatechin gallate, (-)-epicatechin gallate and (-)-epigallocatechin from green tea as well as the mixture of theaflavin and theaflavin gallates from black tea were shown to be the most effective components against the invasion and proliferation of AH109A. These results show that the inhibitory effects of the teas and related components against AH109A cells are due to the cell-specific and higher sensitivity of the cell line to tea components.

Source of ATP for hexokinase-catalyzed glucose phosphorylation in tumor cells: dependence on the rate of oxidative phosphorylation relative to that of extramitochondrial ATP generation

We isolated highly intact and tightly coupled mitochondria from the rat ascites hepatoma cell line AH130 by disruption of the cell membrane by nitrogen cavitation. These isolated mitochondria were found to have essentially the same functional properties as rat liver mitochondria, but unlike the latter, hexokinase (HK) was bound to their membrane. Using the tumor mitochondrial preparation, we examined the source of ATP for phosphorylation of glucose by HK under conditions in which intra- and extramitochondrial ATP-generation systems operated separately or together. Results showed that the membrane-bound HK utilized ATP derived from the most efficiently operating ATP generation system, i.e., oxidative phosphorylation. However, when the rate of extramitochondrial ATP generation was much greater than that of oxidative phosphorylation, HK used ATP from the extramitochondrial ATP-generation system.© 1997 Elsevier Science B.V. All rights reserved.

Assay systems for screening food components that have anti-proliferative and anti-invasive activity to rat ascites hepatoma cells: In vitro and ex vivo effects of green tea extract.

We have developed in vitro and ex vivo assay systems for screening food components and natural substances that suppress the proliferation and/or invasion of a rat ascites hepatoma cell line of AH109A and have used them to study the effect of green tea extract. AH109A cells were found to penetrate underneath the monolayer of primary cultured mesothelial cells isolated from Donryu rat mesentery in the presence of 10% newborn bovine serum. Green tea extract inhibited this AH109A penetration in a dose dependent manner and also inhibited AH109A proliferation in vitro dose-dependently. Green tea tannin, the major polyphenolic substances in green tea extract, also inhibited the proliferation and invasion of AH109A in vitro in a dose dependent manner. When rat serum obtained 0.5 h after oral intubation of green tea extract was added to the culture media instead of newborn bovine serum at a concentration of 10%, the invasion of AH109A was significantly inhibited as compared to control rat serum (before green tea extract intubation); the inhibitory effect lasted for 1 h and disappeared 3 h after oral intubation of green tea extract, but those rat sera showed no inhibition of AH109A proliferation. These results suggest that green tea extract has an inhibitory effect on the invasion of AH109A both in vitro and ex vivo, but on the proliferation of AH109A only in vitro, and that these assay systems are effective for the screening of food components which inhibit tumor cell proliferation and invasion.

Inhibitory effect of serum from rats administered with coffee on the proliferation and invasion of rat ascites hepatoma cells.

The action of coffee on the proliferation and invasion of a rat ascites hepatoma cell line of AH109A was investigated using in vitro and ex vivo assay systems. When rats were given oral intubation of instant coffee powder solution, the sera of those rats had the potent inhibitory activity on both the proliferation and invasion of AH109A. The activity of rat serum was both time- and dose-dependent. The instant coffee powder also inhibited the proliferation and invasion of AH109A in vitro. These results indicate that coffee has anti-proliferative and anti-invasive activity both in vitro and ex vivo. They also suggest that some anti-proliferative and anti-invasive material(s), which may be the ingredient(s) of coffee or their metabolites, appear in rat serum when rats are given oral intubation of coffee, although a possibility that host defense systems may be activated by the oral intubation of coffee cannot be ruled out.

A Revised Structure for the Disialosyl Globo-Series Gangliosides of Human Erythrocytes and Chicken Skeletal Muscle

Disialosyl globo-series gangliosides have previously been isolated from chicken skeletal muscle (E. L. Hogan, R. D. Happel, and J.-L. Chien (1982) Adv. Exp. Med. Biol. 152, 273-278; S. Dasgupta, J.-L. Chien, E. L. Hogan, and H. van Halbeek (1991) J. Lipid Res. 32, 499-506) and human erythrocytes (S. K. Kundu, B. E. Samuelsson, I. Pascher, and D. Marcus (1983) J. Biol. Chem. 258, 13857-13866). In both cases, the structure of this ganglioside was proposed to be NeuAcα2 → 3(NeuAcα2 → 6)Galβ1 → 3GalNAcβ1 → 3Galα1 → 4Galβ1 → 4Glcβ1 → 1Cer (V 3NeuAcV 6NeuAcGb 5Cer). We have reinvestigated the human erythrocyte antigen and now propose an alternative structure differing in the location of the NeuAcα2 → 6 residue: NeuAcα2 → 3Galβ1 → 3(NeuAcα2 → 6)GalNAcβ1 → 3Gal α1 → 4Galβ1 → 4Glcβ1 → 1Cer (V 3NeuAcIV 6NeuAcGb 5Cer). This novel structure is supported by results of 1H-NMR spectroscopy, negative ion fast atom bombardment mass spectrometry, and methylation linkage analysis with capillary gas chromatography-mass spectrometry in both electron impact and chemical ionization modes. Furthermore, based on new results from negative ion fast atom bombardment mass spectrometry and linkage analysis, we propose that the chicken skeletal muscle antigen also has this revised structure, differing only in ceramide composition. The terminal tetrasaccharide of these gangliosides is identical to that of G D1α NeuAcα2 → 3Galβ1 → 3(NeuAca2 → 6)GalNAcβ1 → 4Galβ1 → 4Glcβ1 → 1Cer(IV 3NeuAcIII 6NeuAcGg 4Cer), previously identified in a rat ascites hepatoma cell line (T. Taki, Y. Hirabayashi, H. Ishikawa, S. Ando, K. Ken, Y. Tanaka, and M. Matsumoto (1986) J. Biol. Chem. 261, 3075-3078) and a murine lymphoma cell line with low metastatic potential (K. Murayama, S. B. Levery, V. Schirrmacher, and S. Hakomori (1986) Cancer Res. 46, 1395-1402), although they appear to be immunologically distinct.

Cloning and characterization of a single-stranded DNA binding protein that specifically recognizes deoxycytidine stretch.

We previously identified a G-rich silencer element involved in negative regulation of catalase gene expression in some hepatoma cells (Mol. Cell. Biol., (1992), 12, 2525-2533). To study a nuclear binding protein for this element, we screened cDNA libraries from a rat ascites hepatoma cell line by binding with a synthetic oligonucleotide probe and obtained several clones. One of them, designated SW, was studied in detail. A clone (SW2) of this series contained a near full length cDNA encoding a putative peptide with 463 amino acid residues. We isolated this peptide as a fusion protein. It was found that the protein strongly bound to the C-stretch of the DNA sequence in a single strand specific fashion, but absolutely did not to G-rich sequence. The protein bound weakly to the corresponding double-stranded DNA as well as to C-rich RNA sequence. This protein, though not the expected one, was found to be a novel protein whose DNA binding domain was located on the region containing at least 75 amino acid residues of the carboxyl terminus. A proline rich region was also observed in the middle part of the protein. Northern blot profiles indicated extensive and slight expression of both 2.0 kb and 2.7 kb mRNA species in some hepatoma cell lines and in the rat liver, respectively.

Chemotherapeutic efficacy of the protein-doxorubicin conjugates on multidrug resistant rat hepatoma cell line in vitro

In vitro studies were initiated to study the antitumour effect of protein-doxorubicin (DXR) conjugate on the growth of the multidrug resistant rat ascites hepatoma cell line, AH66DR. The 50% inhibitory concentration (IC50) for DXR in AH66DR cell line was 16 mumol l-1 (AH66 parental cell line, AH66P, IC50 was 0.08 mumol l-1). Treatment of AH66P and AH66DR cells with various concentrations of DXR or conjugates at equivalent concentrations of DXR was performed. The two types of conjugates used were bovine serum albumin (BSA)-DXR conjugate and immunoglobulin G (IgG)-DXR conjugate. Both of these conjugates showed potent dose-dependent inhibition of cell growth against AH66DR cells as compared with the cells treated with DXR or other controls. The IC50 for BSA-DXR and IgG-DXR conjugates in AH66DR cell line was 0.05 (equivalent DXR) mumol l-1 and 0.07 (equivalent DXR) mumol l-1, respectively. These values were similar to that of the AH66P treated with DXR. Cellular uptake and accumulation of DXR or BSA-DXR conjugate was also quantitated in both cell lines. The cellular concentration of DXR in AH66DR cells was 2-fold lower than that of AH66P cells throughout the experiment. In contrast, by the treatment of AH66DR cells with BSA-DXR conjugate, the intracellular drug concentration increased as a function of time up to 24 h (639.1 +/- 41.8, equivalent DXR, ng 10(-5) cells) and reached the same drug level as AH66P cells treated with DXR (617.9 +/- 17.3 ng-5 cells). Ammonium chloride treatment inhibited the effects of the conjugates but did not inhibit the free drugs. Intracellular DXR was effluxed rapidly from AH66DR cells, but BSA-DXR conjugate remained in the cells at relatively high concentration for a long time. These results indicate that by chemically modifying DXR, such as by conjugation of the drug with proteins, it may be possible to overcome multidrug resistance.