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GASTROINTESTINAL, HEPATIC, PULMONARY, AND RENAL

Proapoptotic Effect on Normal and Tumor Intestinal Cells of Cytostatic Drugs with Enterohepatic Organotropism

Department of Physiology and Pharmacology (M.J.M., O.B., J.J.G.M.), Research Unit, University Hospital (M.R.B., M.J.P.), Laboratory of Experimental Hepatology and Drug Targeting, University of Salamanca, Salamanca, Spain

Received March 10, 2005; accepted June 21, 2005.

	Abstract

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The proapoptotic effect of cisplatin bile acid derivatives Bamet-R2 [cis-diamminechloro-cholylglycinate-platinum(II)] and Bamet-UD2 [cis-diammine-bisursodeoxycholate-platinum(II)], developed to treat liver and intestinal tumors, was investigated in vitro using human enterohepatic cells HepG2 (hepatoblastoma), LS 174T (colon adenocarcinoma), and its cisplatin-resistant subline LS 174T/R. Uptake by wild-type tumor cells was higher for Bamets than for cisplatin. In LS 174T/R cells, copper transporter-1 was down-regulated and multidrug resistance-associated protein-2 was up-regulated. Consequently, uptake and efflux of cisplatin, but not those of Bamets, were reduced and increased, respectively. The degree of necrosis (lactate dehydrogenase release) induced by these three drugs was small and similar in all cell types. In contrast, proapoptotic effect (caspase-3 activity and DNA fragmentation) was Bamet-UD2 > cisplatin > Bamet-R2 in HepG2 and LS 174T cells, but Bamet-UD2 > Bamet-R2 >> cisplatin in LS 174T/R cells. This effect was consistent with the ability of these compounds to form DNA-adducts (DNA-platination, changes in the DNA melting temperature, and MspI-induced restriction sequence cleavage). Oral administration of Bamet-UD2 to mice induced mild apoptosis in the small intestine (ileum > duodenum), which was not severe enough to modify its structure or function as determined by water absorption and glycocholic acid uptake by in situ perfused ileum. These results indicate that Bamet-UD2 overcomes the resistance to cisplatin when this is due in part to enhanced ability of intestinal tumors to reduce intracellular cisplatin contents. Moreover, its strong proapoptotic versus its weak pronecrotic effect together with its mild effect on normal tissues, including intestinal mucosa, may account for the high antitumor activity of Bamet-UD2 together with its very low toxicity.

To overcome both drawbacks, i.e., development of resistance by tumor cells and toxicity to normal tissues, a potentially useful strategy is to enhance drug vectoriality toward tumor cells. The aim of this is to reach higher intratumor drug levels, sufficient to carry out their cytostatic effects, while reducing such levels in normal cells. In this line of chemotherapeutic research, cytostatic bile acid derivatives with enterohepatic organotropism named "Bamets" have been developed (for a review, see Marin et al., 2001). Two of the most promising compounds of this family, i.e., Bamet-R2 and Bamet-UD2, have been shown to be efficiently transported by plasma membrane carrier proteins located in enterohepatic epithelial cells, such as transporters for organic anions, cations, and neutral molecules (Briz et al., 2002). This probably explains their ability to accumulate in liver tumor cells and/or be taken up and efficiently secreted into bile by normal hepatocytes (Macias et al., 1998; Larena et al., 2001).

The potential clinical usefulness of Bamets in the treatment of hepatic tumors has been suggested based on their liver organotropism (Macias et al., 1998; Larena et al., 2001), their strong in vitro cytostatic activity, and their in vivo antitumor effect (Dominguez et al., 2001). Interest in these compounds could be extended to the regional treatment of tumors located outside of the enterohepatic circuit. In this respect, a platinated cholic acid derivative, obtained by coupling this bile acid to a carboplatin analog, has been shown to induce apoptosis in a human testicular cancer cell line (Paschke et al., 2003). In these cases, the pharmacological interest of Bamets is assumed to be due to the ability of the liver to efficiently take up and secrete into bile the fraction of the administered drug that may escape from the tumor and reach the general circulation during regional chemotherapy (Macias et al., 1998; Larena et al., 2001).

Beside shared organotropic characteristics, there are important differences regarding the toxic effects on extratumor tissues among different members of the Bamet family, and, more precisely, between Bamet-R2 and Bamet-UD2. In this regard, Bamet-UD2 is particularly interesting because, in addition to its high antitumor activity, no toxicity to the liver, kidney, bone marrow, or nervous system has been detected in in vivo studies (Dominguez et al., 2001). The reason for this difference, although of great interest, is not known.

The aim of the present study was to investigate the contribution of the two ways of cell death, i.e., apoptosis versus necrosis, to the cytostatic effect of Bamet-R2 and Bamet-UD2 on wild-type and cisplatin-resistant enterohepatic tumor cells. This is a relevant issue, since cytostatic activity due to the induction of necrosis may lead to the appearance in vivo of noxious inflammation-associated side effects. Equally interesting is the elucidation of the impact of the proapoptotic effect on normal intestinal cells and how this might affect overall intestinal function, investigated here in mice treated with these drugs. The answer to these questions will be of relevance for developing novel analogs in this family of targeted cytostatic compounds.

	Materials and Methods

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Chemicals. Cisplatin, glycocholic acid sodium salt (GCA), ursodeoxycholic acid (UDCA), Hoechst-33258, propidium iodide, probenecid, RNase A and culture media were obtained from Sigma-Aldrich Laborchemikalien (Seelze, Germany). Specific caspase 3 substrate Ac-DEVD-AMC was from Alexis Corporation (Laüfelfingen, Switzerland). Fetal calf serum and ¹⁴C-radiolabeled GCA (specific activity 56 mCi/mmol) were from Izasa (Madrid, Spain). Low-melting point and normal-melting point agarose were purchased from Invitrogen (Carlsbad, CA). Previously published methods (Criado et al., 1997, 2000) were used to synthesize Bamet-R2 and Bamet-UD2, whose structures are depicted in Fig. 1A. The purity of these compounds was higher than 92% as determined by thin layer chromatography and atomic absorption of platinum. Most of the contaminant material was the natural bile acid used as one of the components of the synthesis. All other chemicals were from Merck Eurolab (Barcelona, Spain).

View larger version (30K): [in this window] [in a new window]   Fig. 1. A, chemical structure of Bamet-R2 and Bamet-UD2. Cytostatic effect of cisplatin, Bamet-R2, or Bamet-UD2, determined by the formazan test as the reduction of living cells after 72 h in culture (B) and drug uptake (C) by wild-type (HepG2 and LS 174T) and cisplatin-resistant (LS 174T/R) cells. Values from formazan test in control non-treated cells (considered as 100%) were compared with those in cells exposed to 50 µM cisplatin, Bamet-R2, or Bamet-UD2 in the absence or the presence of 0.5 mM probenecid (Proben.). Drug contents were measured after incubating cells in the presence of 50 µM cisplatin, Bamet-R2, or Bamet-UD2 for 2 h at 37°C. Drug efflux was investigated in cells previously loaded in the presence of probenecid that were then incubated at 37°C with probenecid-free and cisplatin- or Bamet-UD2-free medium (D). Values are means ± S.E.M. from four different cultures carried out in triplicate. *, p < 0.05 compared with control; , p < 0.05 compared with cisplatin; , p < 0.05 compared with wild-type cells under similar conditions. The Bonferroni method of multiple range testing was used.

Drug Load and Cytostatic Effect. To measure in vitro cytostatic activity, approximately 5000 cells per dish (in 96-well plates) were seeded and the number of living cells was determined by the formazan test (Promega, Madison, WI) after 72 h in culture with 50 µM of the desired compound, as described previously (Larena et al., 2002).

Analysis of steady-state drug uptake by subconfluent cultures was carried out as described previously (Briz et al., 2002). In brief, the cells were incubated in the presence of 50 µM of the desired compound at 37°C for 2 h. Plates were then washed four times with ice-cold serum-free culture medium supplemented with 100 µM cholic acid to reduce the background due to nonspecific binding of bile acid derivatives. Cells were digested with SDS in ultrapure water [0.7% (v/v)]. Platinum contents were determined by flameless atomic absorption spectrophotometry (Briz et al., 2002). Abundance of mRNA for selected carriers was measured by reverse transcription followed by real-time PCR as described previously (Briz et al., 2003) using as gene-specific primers the oligonucleotides whose sequences are shown in Table 1 and SYBR Green I detection, except for ABCC3, for which a TaqMan probe (TGGCCGTGAAGATGCGC) was used.

Biochemical Analyses. Since apoptosis is a dynamic process, to elucidate the most appropriate time to observe it, the appearance of drug-induced DNA fragmentation was investigated in a set of preliminary time-course experiments using LS 174T cells. The cells were exposed to 25, 50, or 100 µM Bamet-UD2 for 14, 24, and 48 h before analyzing DNA-ladder formation as described below. The results of this study (data not shown) recommended the use of 14 h of exposure at 50 µM drug as suitable conditions to carry out the rest of the assays.

As an index of necrosis, lactate dehydrogenase (LDH) activity (Gutmann and Wahlefeld, 1974) released from the cells to the culture medium over 14 h of exposure to the drug under investigation was measured (referred to as LDHout). Cells were digested in lysis buffer containing 50 mM Tris, pH 7.4, 50 mM -glycerophosphate, 15 mM MgCl₂, 15 mM EDTA, 100 µM phenylmethylsulfonyl fluoride, 1 mM dithiothreitol, and 150 µg/ml digitonin for 30 min at 4°C. Cells were then scraped, sonicated, and centrifuged at 200g for 5 min. Then, LDH activity in cell lysates supernatant was measured (referred to as LDHin). The results are expressed as the ratio between LDHout and total LDH activity (LDHout + LDHin). As a positive control of cell death by necrosis, the cells were incubated with 100 mM KCN for 14 h.

To determine caspase 3 activity, after 14 h of exposure to 50 µMof the indicated compound, cell lysates were collected as described above to determine protein concentrations (Markwell et al., 1978) and caspase 3 activity by using a fluorogenic peptide as specific substrate (Hasegawa et al., 1996). Cell lysates (200 µg of supernatant protein) were incubated with assay buffer containing 100 mM HEPES buffer, pH 7.5, 10% sucrose, 10 mM dithiothreitol, and 50 µM of the caspase 3 substrate Ac-DEVD-AMC for varying times at 37°C in a water bath. The reactions were stopped with HClO₄ and centrifuged at 15,000g for 5 min. The fluorescence associated with the release of the hydrolyzed fragment of the peptide was determined fluorometrically.

To further characterize the mechanisms of resistance to cisplatin in LS 174T/R cells, total glutathione (reduced glutathione + oxidized glutathione) contents in trichloroacetic acid supernatants of cell lysates were determined by an enzymatic method. These cell lysates were also used to determine the activity of glutathione S-transferase, as described previously (Perez et al., 2005).

Detection of Apoptosis in Cell Cultures. Subconfluent cultures were exposed for 14 h to the desired concentration of drug dissolved in dimethyl sulfoxide, whose final concentrations in the media were always below 0.2%. Control dishes were treated only with the vehicle. Apoptosis was detected using four different approaches.

One approach was DNA extraction and electrophoresis in agarose gels to assess DNA ladder formation by an adaptation of previously described methods (Benz et al., 1998). Briefly, after drug exposure, culture media were removed and 8 to 10 x 10⁶ cells were incubated in lysis buffer (5 mM Tris-HCl, pH 7.5, 5 mM EDTA, and 0.5% Triton X-100) for 30 min at 4°C. After centrifugation at 20,800g for 15 min, the supernatant fraction was treated with RNase A. DNA was isolated by phenol/chloroform extraction and measured by the PicoGreen fluorometric method (Molecular Probes, Eugene, OR). A fraction of this DNA (600 ng) was electrophoresed in 1.5% agarose gels and visualized by ethidium bromide staining.

A second approach involved staining of DNA with Hoechst-33258 (5 µg/ml; 20 min in the dark) in 4% paraformaldehyde-fixed cells, followed by fluorescence microscopy to visualize condensed chromatin and apoptotic bodies.

The third approach used single cell electrophoresis (comet) assays (Ostling and Johanson, 1984). Briefly, after drug exposure, cells were trypsinized, counted, and suspended (2000 cells/µl) in PBS buffer. An aliquot (10 µl) of cell suspension was carefully mixed with 100 µl of low-melting point agarose [37°C; 0.8% (w/v) in PBS] and placed on a microscope slide precoated with normal-melting point agarose [1% (w/v) in water]. After solidification at 4°C, the slides were submerged in lysis buffer (2.5 M NaCl, 100 mM Na₂EDTA, and 10 mM Tris, supplemented with 1% Triton X-100 and 10% dimethyl sulfoxide just before use) at 4°C for 60 to 90 min. The slides were then placed in a horizontal electrophoresis unit and covered with buffer containing 300 mM NaOH, 1 mM Na₂EDTA (pH >13; 15°C). After 40-min incubation to allow DNA unwinding, alkaline electrophoresis was performed in the same buffer for 20 min at 300 mA. To remove the excess of alkali, the slices were then washed with 0.4 M Tris, pH 7.5, and stained with ethidium bromide to visualize the nuclei.

The fourth approach was DNA analysis by flow cytometry to quantify the percentage of apoptotic cells. After drug exposure, cells were harvested by trypsinization, washed with ice-cold Ca²⁺- and Mg²⁺-free PBS, followed by centrifugation at 500g for 5 min. The pellet was suspended in PBS, fixed with 9 volumes of ice-cold 70% ethanol, and stored at 4°C for 24 h. Cells were then washed twice with PBS and resuspended in PBS, pH 7.8, containing 192 mM Na₂HPO₄, 4 mM citric acid, 1 mg/ml RNase A, and 0.2% Nonidet-P40, incubated at 37°C for 30 min, stained with propidium iodide (33 µg/ml), and analyzed using a FACSort flow cytometer (BD Biosciences, San Jose, CA) and CellQuest software (BD Biosciences).

Detection of DNA-Drug Interactions. The ability to form DNA-adducts was studied using three different approaches. 1) Degree of DNA platination. Platination was measured by flameless atomic absorption spectrophotometry determination of platinum contents in DNA extracted from cells incubated with 50 µM cisplatin or Bamet-UD2 or 100 µM Bamet-R2 for 14 h, as described previously for DNA ladder formation assay. Two additional assays were carried out in a cell-free system using a double-stranded DNA fragment of 1.58 kilobases obtained by PCR using human -actin as the target sequence (from 85 to 1665 base pairs in the accession number BC002409 [GenBank] sequence). DNA was purified by phenol-chloroform extraction followed by isopropanol precipitation. 2) DNA melting temperature. DNA (300 ng) in 25 µl of 10 mM Tris, pH 8.0, containing 50 nM 5-carboxy-X-rhodamine (Molecular Probes), SYBR Green I (1/20,000 diluted; Molecular Probes), and the desired amount of drug was incubated at 37°C for 1 h and then from 60 to 95°C during fluorescence measurement in a ABI Prism 5700 sequence detection system (PerkinElmer Life and Analytical Sciences, Boston, MA). 3) Sensitivity of drug-DNA adducts to endonucleases. The DNA fragment, obtained and preincubated with the desired drug as described above, was incubated in the appropriate buffer with MspI, which recognizes the G-rich restriction sequence GGCC, at 37°C for 2 h before carrying out agarose gel electrophoresis (Bartoszek et al., 2003).

In Vivo Studies. Overnight-fasted adult male CD1 mice (approximately 30 g b.wt.) from the University of Salamanca were used according to the guidelines of the local ethical committee for the use of laboratory animals. The mice received intragastric administration of drugs dissolved in 500 µl of saline or vehicle alone. One set of animals was killed by an overdose of sodium pentobarbital anesthesia 24 h after drug administration before obtaining the small intestine, which was approximately 40 cm in length and was divided into two halves (duodenum-jejunum and jejunum-ileum segments). Part of each fragment was used for the detection of DNA nicking by the terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) technique, using the DeadEndTM fluorometric TUNEL system kit (Promega). Green fluorescence staining indicates a positive reaction. To label all nuclei, propidium iodide (red fluorescence) was used. Following the manufacturer's instructions, as a positive control for the detection of DNA fragmentation, a section from a control intestine treated with DNase I was used. The rest of the intestinal fragment was homogenized to measure caspase 3 activity as described above.

A different set of animals was anesthetized with sodium pentobarbital (i.p., 50 mg/kg b.wt.) 24 h after Bamet-UD2 (30 nmol/g b.wt.) or saline administration to carry out single-pass in situ perfusion of the last 20-cm segment of the small intestine at 150 µl/min with glucose-free Krebs-Henseleit solution (120 mM NaCl, 5 mM KCl, 0.65 mM MgSO₄, 1.17 mM KH₂PO₄, 1.29 mM CaCl₂, and 25 mM NaHCO₃) supplemented with 100 mg/ml gentamicin. After a 30-min equilibrium period, the perfusion medium was replaced by fresh medium containing 50 µM [¹⁴C]GCA, which was perfused for 15 min and then replaced again by [¹⁴C]GCA-free fresh medium. Perfusate samples were collected in preweighed vials at 5-min intervals over 60 min to determine the outflow of perfusate by assuming a density value of 1 mg/µl and to measure radioactivity on a liquid scintillation counter (Beckman Coulter, Fullerton, CA).

Statistical Methods. Results are expressed as means ± S.E.M. To calculate the statistical significance of the differences between groups, Student's t test or the Bonferroni method for multiple range testing was used as appropriate.

View larger version (22K): [in this window] [in a new window]   Fig. 2. Release of LDH to the culture media by wild-type (HepG2 and LS 174T) and cisplatin-resistant (LS 174T/R) cells. Cells were exposed for 14 h to no drug (Control) or 50 µM cisplatin, GCA, Bamet-R2, UDCA, or Bamet-UD2. As a control of intense necrosis, some cells were incubated with 100 mM KCN. LDH activity was measured in both the culture medium (LDHout) and in cell lysates (LDHin). Results are expressed as the ratio of released to total LDH activity: LDHout/(LDHin + LDHout). Values are means ± S.E.M. from three different cultures carried out in triplicate. *, p < 0.05 compared with control by the Bonferroni method of multiple range testing.

View larger version (20K): [in this window] [in a new window]   Fig. 3. Caspase 3 activity in wild-type (HepG2 and LS 174T) and cisplatin-resistant (LS 174T/R) cells. Cells were exposed for 14 h to no drug (control) or 50 µM cisplatin, GCA, Bamet-R2, UDCA, or Bamet-UD2. Caspase 3 activity was measured using a fluorogenic peptide as a specific caspase 3 substrate. Results are expressed as fluorescence arbitrary units (FAU) per hour per milligram of protein. Values are means ± S.E.M. from three different cultures carried out in triplicate. *, p < 0.05 compared with control by the Bonferroni method of multiple range testing.

	Results

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Measurement of the amount of drug accumulated in the cells over 2 h revealed that the amount of Bamet-R2 and Bamet-UD2 found in HepG2 and LS 174T cells was markedly higher than that of cisplatin (Fig. 1C). Drug content in LS 174T/R cells was significantly reduced for cisplatin, but not for Bamet-R2 or Bamet-UD2 (Fig. 1C). Probenecid, a well known inhibitor of several multidrug resistance-associated proteins (MRPs) (Evers et al., 1996), had no effect on cisplatin load in wild-type cells but enhanced that in LS 174T/R cells (Fig. 1C), which resulted in recovery of cisplatin-induced cytostatic effect (Fig. 1B). In contrast, probenecid was without effect on drug load and cytostatic effect of Bamets, either in wild or resistant cells. To evaluate whether cisplatin efflux was enhanced in LS 174T/R cells, after loading the cells in the presence of probenecid for 2 h, the medium was replaced by a fresh drug-free, probenecid-free medium. As shown in Fig. 1D, LS 174T/R cells exported cisplatin at a more rapid rate than LS 174T cells. Efflux of Bamet-UD2 was lower and not markedly different between both cell types. The role of changes in the expression of uptake carriers and efflux pumps was investigated. No change in the expression of import carriers was observed, except for CTR1, the copper importer, that is thought to mediate cisplatin uptake (Safaei and Howell, 2005), which was markedly down-regulated in LS 174T/R cells. Among export pumps, MRP2, which is believed to play a key role in cisplatin resistance (Cui et al., 1999; Leonard et al., 2003), but not P₁-type ATPases ATP7A and ATP7B, which may also mediate cisplatin efflux (Safaei and Howell, 2005), was markedly up-regulated in LS 174T/R cells (Table 2).

Although decreased accumulation, which may be due to defects in uptake and/or enhanced efflux, is the single most commonly observed change in resistant cells, and uptake/export mechanisms seem to be also involved in the resistance of LS 174T/R cells to cisplatin, other typical processes involved in tumor resistance to this drug may contribute to this phenotype. Thus, the amount of total intracellular glutathione was increased from 22.7 ± 1.0 nmol/mg protein (n = 5) in LS 174T cells to 28.2 ± 1.1 nmol/mg protein (n = 5) in LS 174T/R cells (p < 0.01). Moreover, glutathione S-transferase activity was also enhanced from 332 ± 13 nmol of 1-chloro-2,4-dinitrobenzene (CDNB)/min/mg protein (n = 5) in LS 174T cells to 571 ± 32 nmol CDNB/min/mg protein (n = 5) in LS 174T/R cells (p < 0.01).

Necrotic versus Apoptotic Cell Death. Compared with the positive control of necrosis used in the present study, i.e., KCN-induced cell death, a smaller degree of necrosis was observed in all three cells lines assayed (Fig. 2). Treatment with GCA or UDCA had no effect on the release of LDH to the culture media compared with the control cultures. This indicated the absence of cytotoxicity of the bile acid-moieties of Bamet-R2 and Bamet-UD2 molecules, at least at this concentration. In contrast, cisplatin and both Bamets induced a mild but significant increase in the release of LDH activity from the cells into the medium. This was always lower than 5% of the total LDH activity (Fig. 2).

View larger version (58K): [in this window] [in a new window]   Fig. 4. Detection of apoptosis by DNA-ladder formation in human hepatoblastoma HepG2 cells (A), human colon adenocarcinoma LS 174T cells (B), and cisplatin-resistant human colon adenocarcinoma (LS 174T/R) cells (C) cultured in the presence of cisplatin, GCA, Bamet-R2, UDCA, or Bamet-UD2 at the indicated concentrations for 14 h. DNA was extracted, electrophoresed in a 1.5% agarose gel (600 ng of DNA/lane) and stained with ethidium bromide. A representative gel of three similar experiments is shown.

Evidence for Proapoptotic Activity. DNA fragmentation after treatment of HepG2 (Fig. 4A) or LS 174T (Fig. 4B) cells with 50 µM cisplatin or Bamet-UD2 was observed. This effect was less marked in the case of Bamet-R2, even when 2-fold concentrations of this drug were used. No clear signs of DNA fragmentation were observed when cells were incubated in the absence of these drugs or in the presence of their parent bile acids: GCA or UDCA. The ability of Bamet-UD2, and to a lesser extent that of Bamet-R2, to induce DNA fragmentation was also observed in LS 174T/R cells, in which cisplatin-induced DNA fragmentation was not detectable (Fig. 4C).

Using both the comet assay and nuclear staining with Hoescht-33258, apoptosis-associated morphological changes were seen in wild-type cells exposed to cisplatin, Bamet-UD2 and, although to a much lesser extent, in cells treated with Bamet-R2. The proapoptotic effect of Bamet-UD2 and Bamet-R2, but not of cisplatin, on LS 174T/R cells was also confirmed by these two approaches. For the sake of clarity, only the results obtained in cells exposed to Bamet-UD2 are depicted in Fig. 5.

View larger version (83K): [in this window] [in a new window]   Fig. 5. Representative pictures of apoptotic DNA fragmentation detected by tail formation (arrows) in single cell electrophoresis (comet assay) and Hoechst-33258 staining in cells previously fixed with paraformaldehyde (insets) of apoptotic nuclei and bodies (arrowheads) in HepG2 (A and B), LS 174T (C and D), and LS 174T/R (E and F) cells exposed to 50 µM Bamet-UD2 (B, D, and F) for 14 h. Untreated control cells (A, C, and E) are also shown for comparison.

In all three cell lines studied, after single cell electrophoresis a typical comet-pattern, consistent with the presence of nuclear DNA fragments with different migration speeds, and hence that ran for different distances during the electrophoretic process, was observed in Bamet-UD2-treated cells (Fig. 5). By contrast, untreated control cells showed the spherical shape corresponding to undamaged DNA.

Nuclear staining with Hoescht-33258 (Fig. 5, insets) was used to reveal the nuclear morphology typical of apoptosis: chromatin condensation, fragmented DNA and the presence of apoptotic bodies, which was only seen occasionally in untreated control cells or in cells treated with the bile acids GCA or UDCA (data not shown), but which was evident for cells treated with Bamet-UD2 (Fig. 5, insets).

To quantify the extent of DNA fragmentation and formation of apoptotic bodies, flow cytometry analysis was carried out after staining the cells that had been exposed to these drugs with propidium iodide. Figure 6 shows typical histograms depicting counts in the hypodiploid area (M1). The percentage of events in this area is usually considered as an index of the degree of apoptosis (Bai et al., 1999). As shown in Table 3, exposure of HepG2 cells to 50 µM cisplatin, Bamet-R2, or Bamet-UD2 resulted in a moderate but significant increase in the percentage of apoptotic cells in the culture. The proapoptotic effect was more marked in LS 174T cells for cisplatin and Bamet-UD2 (Bamet-UD2 > cisplatin). Basal apoptosis was already enhanced in LS 174T/R cells. This was further increased by Bamet-R2, and more markedly so by Bamet-UD2, but apoptosis was not significantly increased by cisplatin. No significant difference was found between control cells and cells treated with GCA or UDCA.

View larger version (32K): [in this window] [in a new window]   Fig. 6. Representative flow cytometry DNA histographs corresponding to the analysis of DNA content by flow cytometry of LS 174T cells exposed for 14 h to no drug (A; control) or 50 µM cisplatin (B), GCA (C), Bamet-R2 (D), UDCA (E), or Bamet-UD2 (F). Cells were then harvested, fixed in 70% ethanol, and stained with propidium iodide. The zone designated hypodiploid area, or M1 (fragmented apoptotic cells), is depicted on the graphs.

View larger version (40K): [in this window] [in a new window]   Fig. 7. A, detection of DNA-drug interaction by determination of platinum contents in DNA purified from LS 174T and LS 174T/R cells previously exposed to cisplatin, Bamet-R2, or Bamet-UD2 for 14 h. Values are means ± S.E.M. from three different cultures carried out in triplicate. *, p < 0.05 compared with wild-type cells by the Student's t test. B, sensitivity to cleavage by the restriction enzyme MspI of GC-rich restriction sites (GGCC) in a DNA fragment of 1.58 kilobases obtained by PCR from human -actin sequence and previously incubated with 50 µM cisplatin or Bamet-UD2 or 100 µM Bamet-R2 at 37°C for 1 h. C. First negative derivative curves calculated from plotting changes in temperature versus fluorescence (–dF/dT) of SYBR Green I bound to a double-stranded DNA fragment of human -actin mentioned above previously incubated with cisplatin, Bamet-UD2, or Bamet-R2 at 37°C for 1 h. In untreated DNA (control), the peak indicates the DNA melting temperature.

Drug-Induced Apoptosis in Normal Intestinal Cells. A dose-dependent effect of oral administration of cisplatin on caspase 3 activity in mouse small intestine was found (Fig. 8A). Basal caspase activity was higher in duodenum-jejunum compared with jejunum-ileum (Fig. 8B). No significant difference between cisplatin and Bamet-UD2 regarding their effect on both intestinal segments was observed. The proapoptotic effect of both drugs was more marked in jejunum-ileum (Fig. 8B), although the degree of caspase activity did not reach that of the basal activity found in duodenum-jejunum. The results obtained in TUNEL assay were fully consistent with those of caspase 3 activity; moreover, they revealed that most apoptotic cells were located within the intestinal villi rather than in the epithelial layer (Fig. 9).

View larger version (26K): [in this window] [in a new window]   Fig. 8. Caspase 3 activity in small intestine as measured using a fluorogenic peptide as a specific caspase 3 substrate on intestinal homogenates from the first and second half of the whole organ obtained from mice that had received intragastric administration of 500 µl of saline alone or containing the desired amount of drug. A preliminary dose-effect study with cisplatin was carried out (A) to select the dose of 30 nmol/g b.wt. in order to compare the effect of cisplatin and Bamet-UD2 (B). Results are expressed as fluorescence arbitrary units (FAU) per minute per milligram of protein. Values are means ± S.E.M. from measurements carried out in triplicate on five animals per group. *, p < 0.05 compared with control by the Bonferroni method of multiple range testing.

View larger version (105K): [in this window] [in a new window]   Fig. 9. In situ detection of apoptotic nuclei by TUNEL technique in small intestine of mice that had received a intragastric bolus of saline alone (Control) or containing 30 nmol/g b.wt. Bamet-UD2 24 h before tissue sample collection. All nuclei were labeled with propidium iodide (red fluorescence). Immunofluorescent green/yellow staining indicates a positive reaction and hence the presence of DNA nicking. As negative and positive controls of the technique, sections from a control rat were incubated without rTdT (A) or were treated with DNase I to induce extensive DNA fragmentation (B). Representative sections of the first and the second half of the intestine of control (C and E, respectively) and Bamet-UD2-treated (D and F, respectively) animals.

	Discussion

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The antitumor activity of cisplatin is in part due to its ability to form DNA adducts, mainly with the N7 of guanine, which leads to the appearance of both intrastrand and interstrand crosslinks. This, in turn, induces structural distortions in the DNA molecule (Horacek and Drobnik, 1971; Pinto and Lippard, 1985). Like cisplatin, the cytostatic effect of Bamets has also been related to the ability of these compounds to interact with DNA (Marin et al., 1998; Martinez-Diez et al., 2000). Findings of the present study support the notions that Bamet-R2, and, more markedly, Bamet-UD2 are able to form adducts with DNA and that this probably involves guanine moieties. This interaction profoundly affects DNA structure, as indicated by observed shifts in the melting temperature curves. This occurs in such a way that, in proliferating cells, replication cannot be completed (antiproliferative effect) or results in nonviable daughter cells (Marin et al., 1998; Martinez-Diez et al., 2000). The formation of platinum-DNA adducts at both the nuclear and mitochondrial levels is also thought to mediate the cytotoxic effects of cisplatin, and probably also those of Bamets, by inhibiting transcription-dependent cell functions, which may activate intracellular processes that finally end in cell death, in part due to apoptosis (Ormerod et al., 1994). However, changes in treated cells may also induce acute/intense damage, resulting in necrosis. The contribution of these two patterns of cell death during antitumor chemotherapy is of great pharmacological relevance since it may elicit the appearance of inflammation associated side effects.

All the in vitro tests that have been carried out revealed that, at least at the concentrations used in the present study, the natural bile acids used as parent compounds in Bamet-R2 and Bamet-UD2 synthesis, i.e., GCA and UDCA, respectively, did not induce cell death by either necrosis or apoptosis. In contrast, cisplatin, Bamet-R2, and Bamet-UD2 showed strong cytostatic activity, due to a combination of similarly low pronecrotic and marked proapoptotic effects. Even in cells in which cisplatin had partly lost this ability, both Bamets were still able to induce cell death. In addition to this ability, the antiproliferative effects of these drugs due to the formation of DNA-Bamet adducts that inhibit DNA synthesis (Marin et al., 1998; Martinez-Diez et al., 2000) may also have contributed to the reduction in culture size observed after 72-h exposure to these drugs. This may explain why Bamet-UD2-induced inhibition of cell culture growth was similar in LS 174T/R and in LS 174T cells in spite of more marked activation of caspase-3 in LS 174T cells.

Several proapoptotic drugs, including cisplatin, trigger signature-type DNA degradation, resulting in 180-base pair fragments and multiples thereof due to the internucleosomal cleavage carried out by apoptosis-specific endonucleases. This hallmark in apoptosis was clearly observed in Bamet-UD2-treated cells, including LS 174T/R cells, in which cisplatin-induced apoptosis was reduced. Genotoxicity, as evidenced by breaks in nuclear DNA strands, was also detected by single cell electrophoresis or the comet assay. Untreated control cells displayed round nuclei with no comet tails; that is, the DNA remained within the perimeter of the spherical masses, indicating a minimal level of DNA fragmentation. In contrast, cells exposed to DNA-damaging agents, such as UV irradiation and chemical agents, including cisplatin and Bamets as found in the present study, formed nucleoids with "comet-like" tails. This assay has already been used in other studies in the past to detect the cellular DNA damage induced by proapoptotic bile acids such as deoxycholic acid when used at highly toxic concentrations (Powolny et al., 2001). In the present study, at the same relatively low concentration used for Bamet-R2 and Bamet-UD2, GCA and UDCA showed no ability to induce DNA fragmentation. This implies that the ability of Bamets to induce apoptosis was not endowed by their bile acid moieties.

The induction of apoptosis is mediated by activation of complex and interrelated mechanisms at different cell levels. The initial signal can be given at the plasma membrane, involving death receptors. Alternatively, damage at the nuclear or mitochondrial level can trigger apoptosis by only partly shared pathways (Boulikas and Vougiouka, 2003). Whether impairments in these signaling pathways might play a role in cisplatin-resistance is not known. Although differences in gene expression profiles between cisplatin-sensitive and cisplatin-resistant cells have been reported (Briz et al., 2003; Huerta et al., 2003), changes in the expression levels of pro-caspases 3, 8, and 9, which ultimately mediate the degradation of macromolecules in apoptotic cells, have not been reported. In agreement with the concept of a maintained expression of pro-caspase 3, in the present study we have observed that Bamet-UD2 was still able to induce activation of caspase 3 in cisplatin-resistant LS 174T/R cells. However, this occurred less prominently than that induced in the parental cisplatin-sensitive cell line LS 174T.

One important mechanism involved in resistance to antitumor chemotherapy is the overexpression of export pumps, which reduces the intracellular concentrations of many different compounds and hence endows tumor cells with the MDR phenotype. Some of these proteins, including P-glycoprotein or MDR1 (gene symbol ABCB1) and several isoforms of MRP (multidrug-associated protein, gene symbol ABCC) such as MRP1 and MRP2, are expressed in wild-type LS 174T cells and are up-regulated in LS 174T/R cells. The overexpression of export pumps involved in cisplatin resistance, mainly that of MRP2 (Cui et al., 1999; Leonard et al., 2003), could explain the markedly lowered cisplatin load found in these cells compared with wild-type LS 174T cells. P₁-type ATPases, ATP7A and ATP7B, involved in the etiology of Menkes and Wilson's diseases, respectively, due to impaired copper transport, have been suggested to play a role in cisplatin export and hence in some cases, but not all, of resistance to cisplatin (Safaei and Howell, 2005). However, these copper transporters do not seem to be involved in cisplatin-resistance by LS 174T/R cells, because their expression was not enhanced but instead diminished in these cells. Regarding drug uptake, this of Bamets is mainly mediated by several members of the SLC10A, SLCO, and SLC22 families (Briz et al., 2002), whose expression is not impaired in LS 174T/R cells. In contrast, cisplatin uptake is probably carried out by CTR1 or SLC31A1 (Safaei and Howell, 2005), which is down-regulated in LS 174T/R cells and hence may contribute to the reduction of intracellular levels of the drug in these cells. The observation that the development of resistance by LS 174T/R cells was not accompanied by a reduction in Bamet-R2 or Bamet-UD2 loads supports the hypothesis that tumor resistance to cisplatin can be overcome by coupling this cytostatic agent to a bile acid moiety. Moreover, conjugation with glutathione, which plays an important role in cisplatin inactivation by tumor cells, and which indeed seems to be enhanced in LS 174T/R cells, is not an efficient mechanism of resistance against Bamet-R2 or Bamet-UD2, since these drugs do not undergo major biotransformation (Macias et al., 1998; Larena et al., 2001).

The marked enterohepatic organotropic characteristics of Bamet-UD2 (Larena et al., 2001) suggest that it could be administered orally. Moreover, even when administered intravenously, efficient elimination of Bamet-UD2 into bile leads to exposure of the intestinal mucosa to this compound. Therefore, evaluation of the effect of Bamet-UD2 on this tissue is relevant. Although apoptosis was induced to an extent similar to that caused by cisplatin, this did not lead to any apparent functional impairment, at least as indicated by the absence of any change in passive water absorption and active bile acid uptake. Indeed, similar extents of water absorption (17% of the perfused volume) and GCA uptake (62% of the perfused dose) were found in the controls and Bamet-UD2-treated animals.

In conclusion, our findings indicate that Bamets overcome the resistance to cisplatin that is in part associated with an enhanced ability of tumors to reduce the intracellular content of this latter drug. Moreover, they suggest that the strong proapoptotic and weak pronecrotic effect of Bamet-UD2 may account for its previously reported in vivo properties: high antitumor activity and very low toxicity to the liver, kidney, bone marrow, and nervous system (Dominguez et al., 2001).

	Acknowledgements

 
We thank M. I. Hernandez for secretarial help, E. Flores and M. T. Sanchez Montero for technical help, and N. Skinner for English revision of the manuscript.

	Footnotes

 
This study was supported in part by Ministerio de Ciencia y Tecnología, Plan Nacional de Investigación Científica, Desarrollo e Innovación Tecnológica Grant BFI2003-03208, and Junta de Castilla y León Grant SA013/04 (Spain). The group is a member of the Spanish Network for Cooperative Research on Hepatitis, Instituto de Salud Carlos III, Spain (Grant G03/015).

doi:10.1124/jpet.105.086165.

ABBREVIATIONS: MDR/mdr, multidrug resistance; GCA, glycocholic acid; UDCA, ursodeoxycholic acid; Bamet-R2, cis-diamminechlorocholylglycinate-platinum(II); Bamet-UD2, cis-diammine-bisursodeoxycholate-platinum(II); PCR, polymerase chain reaction; LDH, lactate dehydrogenase; PBS, phosphate-buffered saline; TUNEL, terminal deoxynucleotidyltransferase dUTP nick-end labeling; MRP, multidrug resistance-associated protein; CTR1, copper transporter-1; CDNB, 1-chloro-2,4-dinitrobenzene.

Address correspondence to: Dr. Jose J. G. Marin, Department of Physiology and Pharmacology, Campus Miguel de Unamuno, E.D. S-09, 37007-Salamanca, Spain. E-mail: jjgmarin{at}usal.es

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