2016 Volume 64 Issue 11 Pages 1539-1545
6-Mercaptopurine (6-MP) is a clinically important antitumor drug and its commercially available form is provided as monohydrate, belonging to biopharmaceuticals classification system (BCS) class II category. The combination of bismuth(III) (Bi(III)) with 6-MP was proved to significantly improve the anticancer activity of 6-MP, leading to the discovery of a new amorphous complex ([Bi(MP)3(NO3)2]NO3). The prepared [Bi(MP)3(NO3)2]NO3 was characterized by the matrix assisted laser desorption-ionization time-of-flight (MALDI-TOF)-MS, etc. Noticeably, according to the in vitro evaluations of cytotoxicity, cellular apoptotic, colony formation as well as cell migration, the anticancer activity of amorphous [Bi(MP)3(NO3)2]NO3 was found to be of high therapeutic effect over 6-MP.
Currently, in many cases, promising drug candidates have been eliminated from late-stage cancer development due to their poor bioavailability/solubility.1) In order to overcome the solubility limitations and alter the physicochemical, biopharmaceutical, and/or pharmacotechnical characters of a given drug, pharmaceutical scientists generally explore different solid-state forms of a certain active pharmaceutical ingredient (API). Solid drugs could exist in various forms such as amorphous and different polymorphs, solvates, and salts. Most recently, by comparison with the crystalline state of APIs, the amorphous state ones have been found to have higher dissolution rates and better solubility due to the higher degree of free energy. However, the stability usually decreases with increasing free energy. Amorphous solids, therefore, tend to be thermodynamically unstable compared with their crystalline equivalents.2)
6-Mercaptopurine (6-MP) is one of important antimetabolite and antineoplastic APIs in the clinical treatment of human acute lymphoblastic leukemia, systemic lupus erythematosus, rheumatoid arthritis and inflammatory bowel disease,3,4) but has not been applied in lung cancer treatment yet so far. Its commercially available form is provided as monohydrate. 6-MP belongs to biopharmaceuticals classification system (BCS) class II category with low oral bioavailability (about 16%) due to its poor water solubility (0.135 mg/mL),5) which to some extent limits the biomedical application of 6-MP. So efforts should be made to improve the bioavailability/solubility of 6-MP and explore its anticancer effect on certain cancer cell lines, like lung cancer.
Bismuth is well known for its extra-low toxicity and environmentally friendly6) and therefore has been widely selected to replace the traditional triple therapy with bismuth-containing quadruple rescue therapy for the treatment of gastropathy infected by Helicobacter pylori.7–10) Such remarkably low toxicity of bismuth compounds has been attributed to their insolubility in neutral aqueous solutions like biological fluids. The element arsenic in the same main group as bismuth, is known for its anticancer effects in tumor cells through binding to thiols in cysteine residues.11) Recently, many protein-labeling agents have been already explored based on the high affinity of trivalent arsenicals with vicinal dithiols.12–15) Therefore, bismuth(III) (Bi(III)), particularly the organometallic Bi(III) complexes, was assumed to have the same quality of anticancer in this work. In addition, the bioactivity of Bi(III) containing complexes like treatment of a variety of gastrointestinal disorders, antitumor, antimicrobial, and antibacterial activities have been reported.7,9,10,16–19) So, the solubility and bioavailability of 6-MP was expected to be improved by coordination between Bi(III) and 6-MP, forming an amorphous complex.
Herein, we showed the synthesis and characteristic of a new Bi(III)-containing complex derived from 6-MP (described as [Bi(MP)3(NO3)2]NO3) and well explored its in vitro anticancer effects on human lung cancer cells of A549 and H460 by evaluating the biological activities including cytotoxicity, colony formation rate, cell migration rate as well as cellular apoptosis. The obtained results demonstrate that the combination of Bi(III) with 6-MP endowed the newly developed amorphous [Bi(MP)3(NO3)2]NO3 with excellent anticancer activity against lung cancer cells and the solubility and bioavailability of the obtained [Bi(MP)3(NO3)2]NO3 were dramatically improved, compared with that of 6-MP. Therefore, the prepared [Bi(MP)3(NO3)2]NO3 was considered to be potentially used as highly efficacious anticancer agent for the treatment of cancer.
6-MP was first dissolved in ethanol with Bi(NO3)3 solution added in afterwards. Once Bi(NO3)3 solution was dropped in, the Bi(III) and thiol easily formed metal-sulfur bond, leading to the formation of yellow solid, [Bi(MP)3(NO3)2]NO3, as shown in Chart 1. The obtained [Bi(MP)3(NO3)2]NO3 was finally washed with acetone. The solubility of [Bi(MP)3(NO3)2]NO3 was measured to be 1.2 mg/mL, about 8.6 times higher than that of 6-MP (ca. 0.14 mg/mL), as shown in Fig. 1. Additionally, solutions of 6-MP and [Bi(MP)3(NO3)2]NO3 were individually prepared at concentrations of 0.1, 0.5 and 1.0 mg/mL. Clearly, multi-phase solutions were achieved at concentrations of 0.1 and 0.5 mg/mL for 6-MP (inset A), while [Bi(MP)3(NO3)2]NO3 solutions at same concentrations were quite transparent (inset B), verifying the improvement in solubility of [Bi(MP)3(NO3)2]NO3 over 6-MP. The result means the obtained compound could be dissolved much easier in gut and its bioavailability was thus improved. Moreover, the solubility of [Bi(MP)3(NO3)2]NO3 was found better than that of the previously reported Zn containing complex as well,20) demonstrating a better bioavailability.
The energy dispersive spectroscopy (EDS) analysis was accompanied to investigate the molar ratio of Bi(III) to 6-MP, attached to scanning electron microscope (SEM) measurements. The result in Fig. 2 showed a molar ratio of 1 : 3 for Bi(III) to 6-MP in [Bi(MP)3(NO3)2]NO3 complex structure. Since proton of C–SH in 6-MP was easily rearranged in acidic media by converting –SH to C=S, three proton rearranged 6-MP coordinated with one Bi(III) through Bi–S bond. The matrix assisted laser desorption-ionization time-of-flight (MALDI-TOF)-MS was recorded in the positive mode to identify the complex, where a peak at m/z 790.04 indicated the presence of complex as [M]+ (Fig. 3). It means the analyzed molecular weight (MW) of the complex was as predicted. Moreover, the other characterizations of [Bi(MP)3(NO3)2]NO3 structure was accomplished by NMR, Fourier transform (FT)-IR, simultaneous thermal analyses (STA), etc. (see Supplementary materials).
The inhibition abilities of both the obtained compound [Bi(MP)3(NO3)2]NO3 and the starting compounds Bi(NO3)3 and 6-MP against the lung cancer cells of A549 and H460 were investigated individually so as to evaluate the anticancer activity of [Bi(MP)3(NO3)2]NO3. Figure 4 shows that 6-MP alone was inactive as IC50 was over 150 µM, about 2 fold higher than that of Bi(NO3)3 (around 70 µM), while [Bi(MP)3(NO3)2]NO3 exhibited excellent anticancer activity against either A549 or H460 cancer cell with IC50 ranging from 7 to 11 µM, lower than that of K(AuMP)2.21) In order to examine the stability of and cytotoxicity of [Bi(MP)3(NO3)2]NO3 itself before being hydrolyzed to 6-MP, IC50 assay of Bi(III)+6-MP mixture (molar ratio of Bi(III) to 6-MP=1 : 3) was performed. The IC50 values of Bi(III)+6-MP (counted based on Bi(III) content) were 104 and 129 µM for A549 and H460, respectively, which was significantly higher than that of [Bi(MP)3(NO3)2]NO3 (ranging from 7 to 11 µM). The result demonstrated that the obtained [Bi(MP)3(NO3)2]NO3 had good stability and the coordination of Bi(III) with 6-MP endowed [Bi(MP)3(NO3)2]NO3 with excellent anticancer activity. The possible reason was because [Bi(MP)3(NO3)2]NO3 could more easily enter cell owing to the better solubility. Therefore, the coordination of Bi(III) with 6-MP exerted pronounced effect to the anticancer capacity of [Bi(MP)3(NO3)2]NO3 against cells A549 and H460, reflecting on lowest IC50 of [Bi(MP)3(NO3)2]NO3 among three compounds. Both time and dose dependence of [Bi(MP)3(NO3)2]NO3 on the change in inhibition rate of cancer cell with time was tested as well. As shown in Fig. 5, both dose and time dependences of [Bi(MP)3(NO3)2]NO3 were observed for the inhibition against A549 and H460 cells. With more [Bi(MP)3(NO3)2]NO3 used, obvious inhibition was found at the very first beginning of [Bi(MP)3(NO3)2]NO3 treatment (Figs. 5A, B). However, the growth of cancer cells A549 and H460 was not efficiently inhibited after being treated over 24 h when the concentration of [Bi(MP)3(NO3)2]NO3 was less than 10 µM. The stable and excellent inhibition rate of [Bi(MP)3(NO3)2]NO3 against two cancer cells was achieved at the concentration higher than 10 µM, but bio-toxicity would be inevitably avoided when the concentration is too high. Therefore, 10 µM of [Bi(MP)3(NO3)2]NO3 was used as the optimum dosage for the lung cancer cells treatment.
Mixture: Bi(III)+6-MP, molar ratio=1 : 3.
Colony formation assay is an effective method to determine the proliferation capacity of single cell. In vitro single cell proliferation over 6 generations forms clone, and each clone contains at least 50 cells. The cell proliferation capacity of the living environment can be evaluated through colony formation assay. The smaller the colony formation rate is, the stronger the drugs prevent the cell proliferation. As aforementioned in the in vitro cytotoxicity assay, 10 µM [Bi(MP)3(NO3)2]NO3 exhibited the preferable inhibition rate against cancer cells A549 and H460, and thus was used throughout the whole colony formation assay with Bi(NO3)3 and 6-MP at the same concentration for comparison. As seen in Figs. 6A and B, compared with control, colony formation rate higher than 50% was observed for both A549 and H460 after being treated with 6-MP. Bi(NO3)3 showed a very low inhibition rate against colony formation as the colony formation rate was very close to 100%, while the colony formation (less than 15%) of both cancer cells was remarkably inhibited once 10 µM [Bi(MP)3(NO3)2]NO3 was used for treatment. These results verified that the prepared [Bi(MP)3(NO3)2]NO3 owned excellent anti-cell-proliferation activity over either Bi(NO3)3 or 6-MP toward lung cancer cells. The good solubility and bioavailability of [Bi(MP)3(NO3)2]NO3 was considered to make contributions to such high anti-cell-proliferation activity.
Data were shown as the mean±S.D. of five independent experiments.
The cell migration ability can be simply evaluated via cell scratch method. In this method, the cells were incubated with drugs right before the scratch experiments started. It would be determined whether the cell migration occurs by observing if the surrounding cells grow and move forward to the central scratch area. As compared in Figs. 7A and B, the cells of A549 and H460 clearly migrated to the scratch area with treatment time getting longer in dimethyl sulfoxide (DMSO) and Bi(NO3)3 and 6-MP treated experiment groups with the cell migration rate still maintaining over 70% for H549 and 50% for H460 in each group after being treated over 24 h (Figs. 7C, D). However, after the 24 h treatment with [Bi(MP)3(NO3)2]NO3, no obvious cell migration to the scratch area was found for either A549 or H460. These results revealed that the combination of Bi(III) with 6-MP endowed [Bi(MP)3(NO3)2]NO3 pretty high anti-migration activity against lung cancer cells A549 or H460, and would likely weaken in vivo tumor migration. More free [Bi(MP)3(NO3)2]NO3 molecules were available to get into cancer cells due to the improved solubility and thus greatly decreased the activity of cancer cells, leading to the rare migration of A549 or H460 cancer cells to scratch area after a given period treatment.
Data were shown as the mean±S.D. of five independent experiments.
Apoptosis pathways are frequently found in malignant cells, which arise from the complex interplay of genetic aberrations and misregulated death pathways.22) As discussed above, [Bi(MP)3(NO3)2]NO3 was proved to be able to kill A549 and H460 cells predominantly through the induction of apoptosis. As shown in Figs. 8A and B, the results of the Annexin-V–fluorescein isothiocyanate/propidium iodide (FITC/PI) double staining for both A549 and H460 indicate that [Bi(MP)3(NO3)2]NO3 dramatically triggered cellular apoptosis, compared with that from either control or experiment groups treated with Bi(NO3)3 and 6-MP. Similarly, once the lung cancer cells A549 and H460 were incubated with [Bi(MP)3(NO3)2]NO3 followed by Hoechst staining (Fig. 9), the majority of the cells obviously condensed and displayed highly-fluorescent nuclei, a characteristic morphology of cells undergoing apoptosis, and [Bi(MP)3(NO3)2]NO3 induced cell apoptosis by 24.95±4.35% for A549 and 50.06±12.05% for H460 (Figs. 8C, D), respectively, which was much higher than that in either control or Bi(NO3)3 treated group (lower than 1.00%). In addtion, although 6-MP would induce cell cycle arrest and apoptosis,23,24) only an apoptosis population of 5.00% was observed for both A549 and H460 cells after being treated with 6-MP. These results suggested that [Bi(MP)3(NO3)2]NO3 considerably induced apoptotic cell death of A549 and H460 cells. Such remakable apoptosis rate in [Bi(MP)3(NO3)2]NO3 group might benefit from its improved solubility and increased toxicity of Bi(III) owing to the coordination with 6-MP.
In all cases, the cells were exposed to the drug for 24 h at 37°C. FL1: the green channel of Annexin-V−FITC; FL2: the red channel of PI.
The combinaiton of Bi(III) with 6-MP was performed by the reaction of an API without oxygen atoms, 6-MP, with a Bi(III) salt, Bi(NO3)3·5H2O, leading to the formation of amorphous [Bi(MP)3(NO3)2]NO3 with excellent anticancer activity. Also, the solubility of [Bi(MP)3(NO3)2]NO3 was much better than that of 6-MP because of the coordination between Bi(III) and 6-MP, which was considered to be favorable for efficacious cancer treatment. Rare report currently records that, an important clinical anticancer drug, shows obviously desirable curative effect on human lung cancer cells. However, 6-MP combining with Bi(III) displayed much lower micromolar IC50 values in the inhibition of cell viability against the A549 and H460 lung cancer cell lines. Moreover, the solubility of 6-MP was increased after being converted to amorphous complex of [Bi(MP)3(NO3)2]NO3, revealing that such new metal-organic amorphous material provides an alternative viable solid form to improve the solubility of APIs hard to be aqueously disolved.
Bismuth nitratepentahydrate (Bi(NO3)3·5H2O), 6-MP, diethyl ether and ethanol were purchased from Aladdin Reagent Databas Inc. (Shanghai, China) and used as received. All reagents are of analytical grade and used without further purification. All aqueous solutions were prepared with ultrapure water (>18 MΩ). EDS was performed with a TESCAN VEGA 3SBH scanning electron microscopy. 1H- and 13C-NMR spectrum was recorded using an ARX 400 nuclear magnetic resonance spectrometer (Bruker, Germany). FT-IR spectra were obtained with a Spectrophotometer Spectrum 2000. MALDI-TOF-MS data was achieved with AB Sciex 5800 MALDI-TOF/TOF™ System (U.S.A.). STAs were performed using a TA instrument SDT Q600, an-alumina crucible and synthetic air flow.
[Bi(MP)3(NO3)2]NO3A Bi(NO3)3 solution was first prepared by dissolving 0.485 g Bi(NO3)3·5H2O solid (1 mmol) in ethanol with the help of a few drops of nitric acid and then added dropwise into 100 mL ethanol solution containing 0.456 g 6-MP (3 mmol). After being refluxed for 1 h at 85°C under stirring, the resulting solution formed a yellow precipitate. The obtained crude product was then thoroughly washed with acetone and dried in vacuo. [Bi(MP)3(NO3)2]NO3 (C15H12BiN15O9S3, MW=851.53): yellow solid, FT-IR (thin film, neat) νmax 1611, 1388, 1213, 871 cm−1; 1H-NMR (400 MHz, DMSO) δ: 13.81 (s, 1H, –C–NH–), 8.43 (s, 1H, –CH=N–), 8.25 (s, 1H, –CH=N–), 3.64 (m, 1H, –C–NH–). 13C-NMR (151 MHz, DMSO) δ: 170.54 (C6), 150.24 (C4), 144.86 (C2), 144.67 (C8), 129.17 (C5). Anal. (%) Calcd: C, 21.16; H, 1.42; Bi, 24.54; N, 24.67; O, 16.91; S, 11.30. MALDI-TOF-MS (m/z): 790.04 [M]+.
Cell CultureHuman lung cancer cell lines A549 and H460 were maintained in Dulbecco’s modified Eagle’s medium (DMEM) (high glucose) (GIBCO, Invitrogen, U.S.A.) and supplemented with 10% fetal bovine serum (GIBCO), 100 units/mL penicillin and 100 mg/mL streptomycin in a humidified incubator under 5% CO2 at 37°C.
Cytotoxicity AssayThe 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was carried out to evaluate the cytotoxicity. A549 and H460 cells were placed in 96-well plates at a density of 1×104 cells/well and then incubated with the tested compounds. After 24 h incubation, cultures were incubated in 100 µL of the medium containing 10 µL 5 mg/mL MTT solution for 4 h at 37°C. Afterwards, the medium with MTT was removed, and 100 µL of DMSO was added to each well so as to dissolve the formazan. The absorbance at 570 nm was measured with a microplate reader (Bio-Tek ELX800, U.S.A.). The inhibitory percentage of each compound at various concentrations was calculated so as to determine the IC50 value.
In Vitro Inhibition Rate of [Bi(MP)3(NO3)2]NO3A549 and H460 cells were seeded in a 96-well plate at a density of 0.5×104 cells/well at 37°C in 5% CO2. The cells were then incubated in a complete medium containing [Bi(MP)3(NO3)2]NO3 with 5 different concentrations (0, 5, 10, 20, 40 µM) according to IC50, respectively. MTT assay was conducted to evaluate the cell viabilities in each group through 0, 12, 24, 48 and 72 h of incubations.
In Vitro Colony Formation AssayA549 and H460 cells were seeded in a 6-well plate at a density of 400 cells/well at 37°C in 5% CO2. The cells were then incubated in a complete medium containing 1% DMSO and 10 µM of Bi(NO3)3 (shown as Bi in all figures below), 6-MP and [Bi(MP)3(NO3)2]NO3. The colony was counted only if it contained more than 50 cells, and the number of colonies was counted on the 7th day after seeding. Colony formation rate was calculated with the equation below:
 | (1) |
A549 and H460 cells were seeded in a 6-well plate at a density of 3×105 cells/well at 37°C in 5% CO2. After cells formed a cell monolayer, the scratch line was made through the cell monolayer vertically with a 200 µL tip. The culture medium was then sucked out with a pipette and the floating cells left was removed by washing with phosphate buffered saline (PBS) for three times. The cultured cells were incubated in 1% fetal bovine serum medium containing 1% DMSO, and Bi(NO3)3, 6-MP as well as [Bi(MP)3(NO3)2]NO3 with an equivalent concentration of 10 µM at 37°C in 5% CO2, respectively. The cell migration was captured at the time of 0, 6, 12 and 24 h with the corresponding cell-uncovered line width recorded. The cell migration rate was calculated according to the below equation:
 | (2) |
Cell apoptosis was evaluated with an Annexin V-FITC/PI apoptosis detection kit on a BD FACSCalibur Flow Cytometry (U.S.A.) and a Hochest33258 kit on a Nikon TE2000-U Inverted Fluorescence Microscope. Briefly, the A549 and H460 cells were seeded in 12-well plates and treated in a complete medium containing 1% DMSO and 10 µM of Bi(NO3)3, 6-MP or [Bi(MP)3(NO3)2]NO3 for 24 h, followed by the harvesting and staining according to the manufacturer’s protocol. The resulting images were finally collected and data were analyzed on Flowjo 7.6 software (Treestar, Ashland, OR, U.S.A.).
Statistical AnalysisExperimental data were compared using the Student’s t-test. Results obtained were expressed as the mean±standard deviation (S.D.) and considered to be statistically significant when * p<0.05, ** p<0.01 or *** p<0.001.
This work was supported by the National Natural Science Foundation of China (21305090, 21401130), the Shanghai Natural Science Foundation (13ZR1428300), the Innovation Program of Shanghai Municipal Education Commission (14YZ086, 14ZZ139), the State Key Laboratory of Analytical Chemistry for Life Science (SKLACLS1502) and the Fundamental Research Funds for the Central Universities (to Shuang Zhou). The authors greatly appreciated these supports.
The authors declare no conflict of interest.
The online version of this article contains supplementary materials.
