Comparative Analysis of Apoptosis-inducing Activity of Codeine and Codeinone

The following chemicals and reagents were obtained from the indicated companies: RPMI1640 medium, Dulbecco's Modified Eagle Medium (DMEM) (Gibco BRL, Grand Island, NY); fetal bovine serum, (FBS), 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT), riboflavin, phenylmethylsulfonyl fluoride (PMSF) (Sigma Chem. Ind., St. Louis, MO); and dimethyl sulfoxide (DMSO) (Wako Pure Chem. Ind., Osaka, Japan). Morphine hydrochloride and codeine phosphate (codeine) were obtained from Sankyo Pharmaceutical Co. Ltd., Tokyo, Japan. Morphine 6-glucuronide was obtained from Shionogi & Co. Ltd., Osaka, Japan. Codeinone was prepared from codeine according to the literature. 22 

HL-60 cells were cultured in RPMI 1640 medium supplemented with 10% heat-inactivated FBS under a humidified 5% CO²atmosphere. Human mammary gland tumor (MCF7), human lung carcinoma (A549), human periodontal ligament fibroblast (HPLF), human gingival fibroblast (HGF), and human pulp cells (HPC) were cultured in DMEM supplemented with 10% FBS. HPLF, HGF and HPC were prepared from the explants of periodontal ligament, gingival, and pulp of first premolars extracted for orthodontic purposes, after obtaining approval from the Institutional Review Board, Meikai University School of Dentistry. In this study, these cells were used between the fifth and tenth passages. MCF7 was obtained from RIKEN Cell Bank, Tsukuba, Japan. A549 was obtained from Dainippon Pharmaceutical Co., Ltd., Osaka, Japan.

Near confluent cells were incubated for 24 h with 0.1 ml of culture medium containing various concentrations of test samples (96-microwell plate, Falcon, Becton Dickinson and Company, Franklin Lakes, NJ). The viable cell number of HL-60 cells in suspension culture was then determined by trypan blue exclusion (table 1). The viable cell number of other adherent cells and HL-60 cells was determined by the MTT method (fig. 1). In brief, the cells were washed once with phosphate-buffered saline (PBS) (137 mm NaCl, 2.7 mm KCl, 9.6 mm NaH²PO⁴, 1.4 mm K²HPO⁴, pH 7.4) and incubated for 4 h with 0.2 mg/ml MTT in fresh DMEM supplemented with 10% FBS. After removing the medium, cells were lysed with DMSO, and the absorbance at 540 nm (which reflects the relative viable cell number) was measured. The 50% cytotoxic concentration (CC⁵⁰) of test samples was determined from the dose–response curve.

DNA was extracted and applied to 2% agarose gel electrophoresis for detection of DNA fragmentation as described previously. 18DNA isolated from ultraviolet (UV)-irradiated HL-60 cells was run in parallel as markers of oligonucleosomal DNA fragments, together with the DNA size marker.

The extent of DNA fragmentation was quantified by fluorometric determination of DNA recovered from the supernatant of cell lysate. Briefly, cells were lysed in 5 mm Tris-HCl (pH 7.4), 1 mm ethylenediaminetetraacetic acid (EDTA)-0.5% Triton X-100 for 20 min on ice. The lysate and its supernatant obtained after centrifugation at 27,000 g  for 20 min were sonicated and their DNA contents were measured by the diamidinophenylindole (DAPI) method, using fluorescence spectrophotometer (F-4500 HITACHI, Hitachi, Ltd., Tokyo, Japan). The percentage of DNA fragmented was defined as the ratio of DNA content in the supernatant to that of total DNA in the lysate. 23–24 

The appearance of apoptotic and necrotic cells was monitored at the individual cell level by staining with Hoechst (H)-33342 (Molecular Probes, Eugene, OR), and Propidium Iodide (PI) (Sigma Chemical Co., St. Louis, MO). 19HL-60 cells (1 × 10⁵/ml) were incubated without or with codeinone for 6 h. At the end of the incubation period, (H)-33342 (final concentration, 1 μg/ml) was added and incubated for 10 min at 37°C. Cells were placed on ice and PI (final concentration, 1 μg/ml) was added to each well. Cells were incubated with dyes for 10 min on ice, protected from light, and then examined under ultraviolet light with the use of a Hoechst filter (Nikon, Melville, NY).

Cells were incubated with test samples for 4 h. Cells were collected by centrifugation at 1,000 g  for 5 min at room temperature, and washed twice in cold PBS. A PNIM3546 ANNEXIN V-VITC Kit (Bechman Coulter Company, Marseille, France) was used for FACS (Becton, Dickinson and Company, Franklin Lakes, NJ). 25,26Cells were added 1 μl Annexin V-FITC and 10 μl PI, and incubated in the dark for 15 min at room temperature. The stained samples were added 400 μl 1× binding buffer and processed by FACS. Cells that have bound Annexin V but do not take up PI (bottom right quadrant, fig. 4) are early apoptotic. Annexin V positive cells that take up PI are late apoptotic (top right quadrant). Annexin negative cells that take up PI are necrotic (top left quadrant). Cells that are negative for both Annexin V and PI (bottom left quadrant) are nonapoptotic and nonnecrotic viable cells.

The cell pellets were resuspended in 100 μl of homogenizing buffer (10 mm Tris-HCl, pH 7.6, 150 mm NaCl, 5 mm EDTA and 2 mm PMSF), and disrupted by passing through a 21.5 gauge syringe 10 times (for the cytochrome c release assay with intact mitochondrial system) or by three cycles of freezing (at −80°C and thawing [on ice] for the caspase 3 degradation assay with disrupted cell system). 27We did not add Trixton X-100 (1%) in the homogenizing buffer to avoid the leak of these proteins from mitochondria. The cell homogenates were centrifuged at 10,000 rpm, 5 min at 4°C to pellet down the mitochondria, and nuclear fraction, and the supernatant was collected. Cytosol fraction was obtained as supernatant after centrifugation of post-mitochondrial supernatant at 105,000 g  15 min from the supernatant. The protein concentrations were measured using a Protein Assay Kit (Bio Rad, Hercules, CA). The equal amounts of the protein (20 μg) were mixed with 2× sodium dodecyl sulfate (SDS)-sample buffer (0.1 M Tris-HCl, pH 6.8, 20% glycerol, 4% SDS, 0.01% bromphenol blue, 1.2% 2-mercaptoethanol), boiled for 10 min, and the aliquots equivalent to 20 μg protein were applied to the 15% SDS polyacrylamide gel electrophoresis, and then transferred to nitro cellulose membranes. Then membranes were blocked with 5% skim milk in Tris-HCl buffered saline plus 0.05% Tween 20 for 1.5 h and incubated with antibodies against cytochrome c (BD Biosciences, Pharmingen, San Diego, CA), antibodies against cytochrome oxidase subunit II (Molecular Probes, Inc., Eugene, OR), and caspase-3 (Transduction Laboratories, Lexington, KY) 1:1000 for 1.5 h at room temperature or overnight at 4°C, and then incubated with horseradish peroxidase-conjugated antirabbit immunoglobulin G (IgG) for 30 min at room temperature. Immunoblots were developed with a Lumi-Light^PLUSwestern blotting substrate (Roche, Molecular Biochemicals, Germany), according to the manufacturer's instructions.

Intracellular caspase 3–like activity was measured using the PhiPhiLuxG¹D²(OncoImmunin, Gaithersburg, MD). 28HL-60 cells (5 × 10⁵/ml) were treated for 30 min or 1 h at 37°C with 34 μm codeinone. Cells were pelleted and cultured for 1 h at 37°C in 75 μl of 10 μm PhiPhiLuxG¹D²substrate. After washing, caspase 3–like activity was measured as follows. Cells were gated to eliminate the PI-stained cells according to the protocol, and the fluorescence intensity in 1 × 10⁴cells were measured at the Fluorescence 1 (FL1) channel. Cells that were not stained were used to set the threshold point.

MnSOD and Cu/ZnSOD activities were measured by activity staining with nitro blue tetrazolium (NBT) (Sigma Chemical Co., St. Louis, MO) and riboflavin directly on the gel after separation on polyacrylamide gel electrophoresis. 29In brief, cells were washed once with cold PBS, dissolved in the sample buffer (1% Triton X-100, 0.25 M sucrose, 10 mm Tris-HCl, pH 7.4) and applied to 9% polyacrylamide gel electrophoresis (45 min, 20 mA). The gel was stained with 2 mg/ml NBT in water for 15 min in the dark, replaced with 10 μg/ml riboflavin in the dark for 15 min, and then illuminated overnight. During illumination, the gel became uniformly blue except for the position containing SOD. The gel was destained by washing in water, and subjected to image processing by scanner (Canoscan N656U, Canon Inc., Tokyo, Japan).

Total RNA was isolated using a Purescript RNA Isolation kit (Gentra systems, Minneapolis, MN) protocol. HL-60 cells (1 × 10⁶/ml) were lysed in 300 μl cell lysis solution, then 100 μl Protein-DNA precipitation solution was added. Lysis fluids were centrifuged at 15,000 g  for 3 min. The supernatant was added 300 μl isopropanol. After centrifugation at 15,000 g  for 3 min, the pellet was washed in 300 μl 75% ethanol. After centrifugation at 15,000 g  for 1 min, the pellet was air dried for 15 min and dissolved in diethyl pyorcarbonate (DEPC)-treated H²O. A reverse transcriptase-polymerase chain reaction (RT-PCR) was performed with 1.0 μg of total RNA using the Rever Tra Ace (Toyobo Co., LTD, Osaka, Japan). Single strand cDNA obtained by RT reaction with oligo (dT)²⁰primer was amplified using the KOD plus (Toyobo Co., LTD, Osaka, Japan), using MnSOD specific primers of (5′-TCCCCGACCTGCCCTACGAC-3′ and 5′-CATTCTCCCAGTTGATTACAT-3′), and Cu/ZnSOD specific primers (5′-ATGGCCACGAAGGCCGTGTGC-3′ and 5′-GGAATGTTTATTG-GGCGATCCCA-3′), G3PDH specific primers (5′–TCCACCACCCTGTTGCTGTA-3′ and 5′–ACCACAGTCCATGCCATCAC-3′), according to the protocol. The RT-PCR products were applied to 2% agarose gel, and the ethidium bromide-stained gel was then photographed under UV light.

All data in this study were expressed as mean ± SE. To compare values between multiple groups in DAPI method, analysis of variance (ANOVA) was applied and a Least Significant Difference Method was used to calculate a P  value. Statistical significance was defined as P  less than 0.01.

The appearance of apoptotic cells and caspase 3–like activity were compared between control and the groups using the Student t  test. Statistically significant difference was assumed if the P  value was less than 0.05.

Codeinone dose-dependently reduced the viable cell number of HL-60 cells (fig. 1). The cytotoxic activity of codeinone in HL-60 (CC⁵⁰= 4.7 ± 0.1 μm) was more than 65 times higher than that of codeine (CC⁵⁰> 325 μm) (table 1). Codeinone induced various apoptosis-associated characteristics in HL-60 cells. Morphological observation with H-33342 staining demonstrated that codeinone produced significant amounts of apoptotic bodies with fragmented nuclei (fig. 2). Agarose gel electrophoresis demonstrated that codeinone induced internucleosomal DNA fragmentation (fig. 3), confirming our previous report. Codeinone, but not codeine, dose-dependently induced DNA fragmentation, as judged by the accumulation of DNA fragments (recovered in the supernatant fraction after centrifugation) (table 2).

FACS analysis demonstrated that codeinone increased the production of apoptotic cells, detected by Annexin staining, from 5.0 ± 2.2% (control, n = 7) to 41.5 ± 5.7% (codeinone-treated;P < 0.001, n = 4) (One of the representative data are shown in fig. 4). It is also notable that codeinone produced small numbers of late apoptotic (top right quadrant, fig 4) and necrotic cells (top left quadrant, fig. 4) with PI. We found that codeine (6450 μm) did not significantly increase the number of apoptotic cells (data not shown). It is well known that DNA fragmentation is executed by caspase-activated DNase (CAD), which has to be first activated by cleavage of inhibitor of CAD by caspase 3. 30,31We confirmed that this actually occurred. Codeinone (34 μm) activated the caspase 3–like activity shortly after treatment (fig. 5). Our repeated experiments further confirmed that the increase of caspase 3–like activity was transient, peaked at 30 min after codeinone treatment (9.8 ± 1.1%;P < 0.05, n = 4), and declined at 1 h, returning to control level (2.6 ± 0.5%versus  2.5 ± 0.4%[control], not significant, n = 4). Caspase activation (fig. 5) was followed by the cleavage of caspase 3 (bottom panel, figure 6). It remains to investigate whether caspase 3 was activated via  mitochondrial pathway or via  caspase 8 independently of mitochondria. 32–34We next examined whether codeinone can induce the cytochrome c release from mitochondria. We found that codeinone actually induced the cytochrome c release, at 34 μm, but it also induced the release of cytochrome oxidase, a mitochondrial enzyme, at a slightly lower concentration (fig. 6). This indicates some mitochondrial damage.

We next investigated whether codeinone modified the activity of mitochondrial MnSOD. When HL-60 cells were incubated for 30 min with lower concentrations of codeinone, the MnSOD activity was slightly reduced. The MnSOD activity returned to the control level at higher concentrations of codeinone. We reproducibly found these concentration-dependent changes of MnSOD activity in a total of four independent experiments (data not shown). On the other hand, codeine did not significantly change the MnSOD activity. However, in contrast to the minor change in MnSOD activity, codeinone did not significantly affect the MnSOD mRNA expression (data not shown).

The current study demonstrates that codeinone, but not codeine, induces apoptosis in HL-60 cells, as judged by several biochemical criteria of apoptosis. The Annexin staining experiment confirmed that codeinone actually induces apoptosis at the individual cell level. However, we unexpectedly found that codeinone produces small proportions of necrotic cells as well as apoptotic cell populations [H-33342 staining (fig. 2) and FACS analysis (fig. 4)], and released cytochrome oxidase into cytosol. These data suggest that codeinone may induce the mitochondrial damage, possibly caused by the induction of necrosis. There remains to investigate whether both apoptotic and necrotic characteristics are expressed in the same cell, or codeinone produces a mixture of apoptotic and necrotic cell populations. Similarly, vitamin K analog induced both apoptosis and necrosis in HL-60 cells. 35Compared with morphine (CC⁵⁰= 1,359–6,552 μm), morphine 6-glucuronide (CC⁵⁰= 424–4,610 μm) (table 1), morphine 3-glucuronide (CC⁵⁰= 3,900–5,200 μm), 10codeinone showed much higher cytotoxicity (CC⁵⁰= 4.7–16.5 μm). Furthermore, codeinone showed some tumor-specific cytotoxic action (table 1). It has been reported that when humans were orally administered 30 mg codeine, the plasma concentration of codeine reached 46 ng/ml after 30–120 min. 36When codeine (60 mg) was orally administered, the maximal plasma concentrations after 1.2 h was 88.1 ng/ml. 37The plasma concentration of codeine is considered to be much lower than that required for apoptosis-induction. This suggests that it is important to establish an efficient delivery system of codeinone for the clinical application. We found that codeinone failed to stimulate both activity and mRNA expression of MnSOD in contrast to morphine. 19This suggests that the apoptosis induction by codeinone is not coupled with the changes in the activity and expression of MnSOD, which is a rescue enzyme for oxidative stress. This is supported by our observation that the cytotoxic activity of codeinone was not significantly affected by various antioxidants other than NAC. 18The reason why the cytotoxic activity was significantly reduced by NAC may be explained by the covalent binding of NAC to α,β-unsaturated ketone moiety in codeinone via  the Michael reaction. Similarly, the inactivation of codeinone by glutathione, a popular cysteine-containing antioxidant, has been reported. 16–17The log P  values of morphine (log P = 0.24), codeine (log P = 0.82), and codeinone (log P = 0.78) were calculated by CLOGP (Pomana College Medical Chemistry Project, Clermont, CA). This demonstrates that higher cytotoxic activity of codeinone, compared with codeine, was not simply caused by the difference in hydrophobicity. Further studies are required to elucidate the mechanism by which codeinone induces the apoptosis/necrosis, cytochrome c release, caspase activation, and finally leads to the internucleosomal DNA fragmentation by the activated DNase, as well as to clarify which pathway, either extrinsic or intrinsic, 38codeinone triggers by investigating the expression of more critical apoptosis markers such as caspase 8, 9, Bax, and Bcl.

Codeinone has been reported to display comparable antinociceptive activity with codeine in mice. 13It has also been evidenced by the tail flick test in rats that codeinone has antinociceptive activity, consistent with a previous report that methoclocinnamox, a novel codeinone was fully effective in warm-water tail-withdrawal assay in rhesus monkeys. 39We have demonstrated for the first time that codeinone shows tumor-specific cytotoxic activity by induction of apoptosis and/or necrosis, and its activity exceeds those of morphine, morphine 6-glucuronide, and codeine. This property of codeinone further adds a possible chemotherapeutic efficacy to be used in combination treatments with other anticancer drugs for patients being treated for cancer pain. 40–41