European Journal of Medicinal Chemistry 96 (2015) 187e195
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Original article
Cytotoxicity profile of novel sterically hindered platinum(II) complexes with (1R,2R)-N1,N2-dibutyl-1,2-diaminocyclohexane Haiyan Zhang a, Shaohua Gou a, b, *, Jian Zhao a, Feihong Chen a, b, Gang Xu a, b, Xia Liu c, ** a
Pharmaceutical Research Center and School of Chemistry and Chemical Engineering, Southeast University, Nanjing 211189, China Jiangsu Province Hi-Tech Key Laboratory for Bio-medical Research, Southeast University, Nanjing 211189, China c Department of Science and Technology, Jiangsu Open University, Nanjing 210036, China b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 7 November 2014 Received in revised form 6 April 2015 Accepted 7 April 2015 Available online 8 April 2015
Four Pt(II) complexes of (1R,2R)-N1,N2-dibutyl-1,2-diaminocyclohexane with two alkyl branches as steric hindrance have been designed and synthesized. In vitro cytotoxicity of these compounds indicated complex 4 is a cytotoxic agent more potent than its parent molecule, oxaliplatin, against almost all the tested cell lines. Agarose gel electrophoresis study showed that the kinetic reactivity of complex 4 with DNA is slow down due to the sterically hindered effect, demonstrating that it may possess a different mechanism of action from cisplatin. Flow cytometry results revealed that complex 4 induced apoptosis of tumor cells by blocking the cell-cycle progression in the G2/M phase. Western blot analysis showed it had a similar apoptotic mechanism to cisplatin which could induce apoptosis via a mitochondrialdependent pathway. © 2015 Elsevier Masson SAS. All rights reserved.
Keywords: Platinum (II) complexes Steric hindrance Potent cytotoxicity Apoptotic mechanism
1. Introduction Cisplatin and its analogs are one of the most important classes of chemotherapeutic agents available for the treatment of solid tumors [1e4]. However, the severe toxic side-effects and insurmountable cross-resistance are the major drawbacks of the present platinum-based drugs. It has been demonstrated that sulfurcontaining biomolecules such as glutathione and metallothionein have a high affinity for PtII complexes [5e8], which are responsible for the side effect and cross-resistance of the platinum(II) complexes. Moreover, the interaction of platinum(II) complexes with sulfur-containing molecules may deactivate the anticancer PtII complexes before the platinum complexes bind to DNA and form effective intrastrand Pt-DNA adducts [9]. Oxaliplatin as one of the best sale platinum based antitumor drugs has been found not to be affected in some cisplatin- and carboplatin-resistance tumors [10e12], this is believed to be largely attributed to its carrier ligand 1R,2R-diaminocyclohexane (1R,2R-DACH) which plays an important role in improving the characteristics of oxaliplatin by increasing lipophilicity and DNA mismatch repair inhibition [11,12].
* Corresponding author. Pharmaceutical Research Center and School of Chemistry and Chemical Engineering, Southeast University, Nanjing 211189, China. ** Corresponding author. E-mail addresses: [email protected], [email protected] (S. Gou). http://dx.doi.org/10.1016/j.ejmech.2015.04.019 0223-5234/© 2015 Elsevier Masson SAS. All rights reserved.
Encouraged by the successful application of 1R,2R-DACH in oxaliplatin, numerous 1,2-DACH derivatives have been designed and introduced to platinum complexes for antitumor study [13e18]. Among them, a small number of symmetric N1,N2-disubstituted 1,2-DACH derivatives have been used as carrier ligands for the improvement of the pharmacological profile of the present platinum based drugs [16e18]. But those ligands reported were not particular designed for increasing the steric hindrance of the resulting platinum(II) complexes to overcome the cross-resistance of the platinum(II) drugs. So far, much effort has been made to increase the steric hindrance of antitumor platinum(II) complexes [19e21], and some of approaches seem to have achieved the ideal results. For example, ZD0473 was synthesized by the introduction of 2-methylpyridine as a carrier ligand, which enables the platinum(II) complex to show considerable cytotoxicity against cancer cell lines, especially against cisplatin-resistant cancer cells with elevated glutathione and/or metallothionein levels due to the steric hindrance of 2methylpyridine [22,23]. Recently, our group has designed a series of platinum-based complexes bearing N-monosubstituted 1R,2Rdiaminocycloohexane derivatives as carrier ligands, aiming to enhance the sterically hindered effect of the resulting complexes [24,25]. The results indicated that N-monoalkyl derivatives of 1R,2R-DACH could effectively improve the pharmaceutical properties of the resulting platinum compounds as compared with
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oxaliplatin [26e28]. According to our previous study, we assumed that the steric hindrance of the substituted group may change the interaction mode between the platinum complexes and DNA, which may subsequently affect antitumor properties of the resulting complexes, thus leading to the absence of the cross resistance with cisplatin or oxaliplatin [27]. It is expected that the increased steric hindrance of platinum complexes can decrease the interaction between platinum complexes and sulfur-containing molecules and prolong time of blood cycle, resulting in enhancing curative effect for tumor. However, it should be kept in mind that huge steric hindrance with the designed platinum complexes may disturb and even prevent the interaction of the compound with DNA. As we believe that the moderate increase of sterically hindered effect of the platinum complexes can improve the cytotoxic activity and decrease the side effects of the present platinum-based drugs, we intend to introduce two alkyl moieties to the 1R,2R-DACH skeleton so as to obtain the leading platinum complexes which can take advantage of the successful leaving ligands like chloride ion in cisplatin, 1,1-cyclobutanedicarboxylate in carboplatin, malonate in heptaplatin and oxalate in oxaliplatin. Herein reported are four novel platinum complexes of (1R,2R)-N1,N2-dibutyl-1,2-DACH as a carrier ligands (Fig. 1) with their antitumor activity as well as tentative study on their mechanism of action.
2. Results and discussion 2.1. Chemistry Platinum(II) complexes were simply prepared in one or two steps following the procedures shown in Scheme 1. The interaction of the ligand (L) with potassium tetrachloroplatinate(II) led to the generation of complex 1. The reaction took a long time than expected, indicating that alkyl species have caused hindrance for the ligand to bind the metal atom. Further reaction of complex 1 with the corresponding silver dicarboxylate in water formed (1R,2R)N1,N2-dibutyl-1,2-DACH -Pt(II) conjugates (complexes 2e4), respectively. The newly synthesized platinum(II) complexes were characterized by microanalysis, IR, 1H and 13C NMR spectra with electrospray ionization mass (ESI-MS) spectroscopy. Complexes 1 and 4 were specially characterized by 195Pt NMR spectroscopy. In the infrared spectra, the NeH stretching vibrations of complexes 1e4 were obviously shifted to lower frequencies than those of free ligand [29], due to the amino group coordination with Pt(II) ions. Besides, the C]O vibration of complexes 2e4 appeared in the range from 1596 to 1698 cm1, characteristic of coordinated dicarboxylates, while the CeO feature appeared in the range of 1310e1356 cm1.
All the platinum(II) complexes show 100% of [MþNaþ] or [MþHþ] peaks, remarkably acompanied with three isotopes of platinum element, 194Pt (33%), 195Pt (34%), and 196Pt (25%), in their positive ESI-MS spectra. In the 1H NMR spectra, the signals of CH2 and CH protons next to the amino groups occurred in the range of 1.55e2.74 ppm and 2.53e3.17 ppm as multiplets, shifting to highfield relative to the corresponding signals (3.64e3.73 and 3.55e3.57 ppm) of the free ligand [29]. The broad signals of hydrogen atoms belonging to amino groups appeared in the range of 5.45e6.27 ppm due to the coordination with the metal atom, shifting to high-field compared with the metal-free ligand [29]. In the 195Pt spectra, both complexes 1 and 4 showed a single peak around 2000 ppm. Furthermore, the chemical shifts of 1H NMR and the numbers of carbon atoms of 13C NMR spectra for complexes 1e4 were in conformity with the proposed molecular structure of the compounds.
2.2. In vitro cytotoxic activity The cytotoxicity of the synthesized complexes was evaluated by MTT assays against human hepatocellular carcinoma cell line (HepG-2), gastric carcinoma cell line (SGC-7901), non-small-cell lung cancer cell line (A549), and colorectal cancer cell line (HCT116). Cisplatin and oxaliplatin were used as positive controls. The corresponding IC50 values are presented in Table 1. It is of much surprise to note that complex 1 had negligible cytotoxicity against all the tested cell lines, although chloride anions seemed to be easily separated from the Pt(II) ion as a leaving ligand in comparison to those dicarboxylates that served as bidentate ligands to bind the metal atom by chelation. Interestingly, both complexes 2 and 3 showed selective activity against certain tested cell lines. The former was sensitive to A549 and HCT-116 cell lines with IC50 values comparable to cisplatin and oxaliplatin, while the latter only expressed activity toward HepG-2 cell line with an IC50 value junior to the positive controls. However, complex 4 showed extraordinary cytotoxicity against all the test cell lines, which notably showed high cytotoxicity superior to that of cisplatin or oxaliplatin against HepG-2, A549 and HCT-116 cell lines except SGC-7901. Especially, complex 4 was 2.6-fold as potent as oxaliplatin toward human colorectal cancer cell line (HCT-116) that is the most sensitive to oxaliplatin among the present platinum based drugs. By comparing the cytotoxicity of complexes 1e4 and positive controls, it is rational to conclude that the existence of alkyl moieties, as steric hindrance, on each amino group of 1,2-DACH has a significant influence on the antitumor property of the resulting platinum complexes in contrast to its parent compound, oxaliplatin. Nevertheless, the role of the leaving ligands on the cytotoxicity of
Fig. 1. Related antitumor Pt(II) complexes in this paper.
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Scheme 1. Preparation of complexes 1e4.
moieties to the 1R,2R-DACH skeleton is an effective way to improve the antitumor activity of the resulting platinum(II) complex by increasing the steric hindrance in complex 4.
Table 1 In vitro cytotoxicity of complexes 1e4, cisplatin and oxaliplatin. Complexes
1 2 3 4 Cisplatin Oxaliplatin
IC50 (mM)a HepG-2
SGC-7901
A549
HCT-116
>100 >100 20.3 ± 1.56 6.5 ± 0.55 7.2 ± 0.84 6.6 ± 0.56
>100 >100 >100 16.9 ± 1.58 2.5 ± 0.23 4.2 ± 0.35
>100 28.3 ± 2.34 >100 8.5 ± 0.76 13.3 ± 0.12 12.5 ± 1.13
>100 30.1 ± 2.85 >100 7.3 ± 0.66 29.3 ± 0.24 19.1 ± 1.89
a Mean value ± standard deviation from three independent experiments; IC50 defined as the drug concentration required to inhibit 50% of cell growth by the MTT assay after 48 h drug exposure.
the newly synthesized platinum compounds can not be ignored. It appears that 1,1-cyclobutanedicarboxylate is a suitable leaving group for the platinum complex with (1R,2R)-N1,N2-dibutyl-1,2diaminocyclohexane as a carrier ligand. Considering complex 4 with much potent cytotoxicity in the preliminary assays, we further investigated its cytotoxicity against the other human cancer cell lines, including breast carcinoma cell lines (MCF-7, MDA-MB-231), gastric carcinoma cell line (BGC-823), leukemia cell lines (K562, NB4), prostatic cancer cell line (DU145), and ovarian cancer cell line (A2780) (Fig. 2). The corresponding IC50 data and those of cisplatin, oxaliplatin and carboplatin as positive controls are listed in Table 2. As expected, complex 4 showed potent cytotoxicity against all the tested cell lines. Notably, it even exhibited superior cytotoxicity to cisplatin in MCF-7, MDA-MB-231, K562, DU145 and A2780 cell lines. In addition, complex 4 was more potent than its parent molecule, oxaliplatin, against almost all the tested cell lines, indicating that it is a potent cytotoxic agent that possesses a broad antitumor spectrum of activity. It is of much interest to note that carboplatin and complex 4 have the same leaving group of 1,1-cyclobutanedicarboxylate, but the biological activity of carboplatin is much weaker than that of our compound. Again, the results indicated that the introduction of two alkyl
2.3. Flow cytometry study 2.3.1. Cell cycle analysis It is generally accepted that the cell cycle arresting induced by a platinum compound is necessary because it enables the nucleotide excision repair (NER) complex to remove the adducts and promote cell survival. When repair is incomplete, the cells will die by apoptosis. For instance, cisplatin inhibits cancer cell growth by blocking the cell cycle through the S or G2/M phase [30,31]. In order to preliminarily reveal the mechanism of action of the synthesized complexes, flow cytometric analysis was applied to detect the inhibitory effect of complex 4 on HCT-116 cell cycle progression. The results are shown in Fig. 3. Obviously, this compound induced S phase cell depletion, suggesting that apoptosis was launched from the S phase, and an accumulation of cells in the G2/M phase was observed as compared with the control tumor cells, which is in accordance with the effect of most anticancer agents. Moreover, the percentage of G2/M phase increased with the concentration of complex 4 increasing from 5 to 20 mM. In general, the compound appeared to induce apoptosis in a dose-dependent manner in HCT-116 cells by the increase of cell population at G2/ M phase. 2.3.2. Apoptosis analysis With the purpose of detecting in which way complex 4 induced cellular death (necrosis or apoptosis), the apoptotic analysis of complex 4, cisplatin and oxaliplatin against HCT-116 and HepG-2 cells was performed by using an Annexin V-FITC/PI assay. The tested compounds were incubated with tumor cells (HCT-116 and
Table 2 Further in vitro assay of complex 4 with cisplatin, oxaliplatin, carboplatin as positive controls. Cell lines
IC50 (mM)f Cisplatin
a
MCF-7 MDA-MB-231a BGC-823b K562c NB4c DU145d A2780e a
± ± ± ± ± ± ±
3.56 1.72 0.51 1.08 0.13 2.35 1.54
25.71 26.82 7.91 39.92 52.82 20.40 6.58
± ± ± ± ± ± ±
1.67 2.25 0.65 2.65 3.76 1.86 0.69
Carboplatin
Complex 4
109.47 ± 8.76 112.26 ± 8.76 43.21 ± 2.54 n.d. 12.59 ± 2.43 n.d.g 17.20 ± 2.41
17.52 13.79 8.28 4.51 9.68 5.05 4.55
± ± ± ± ± ± ±
1.67 5.89 0.74 0.32 0.15 0.43 0.33
Human breast carcinoma cell lines. Human gastric carcinoma cell line. Leukemia cell lines(K562,NB4). d Human prostatic cancer cell line. e Human ovarian cancer cell line. f Mean value ± Standard Deviation from three independent experiments; IC50 defined as the drug concentration required to inhibit 50% of cell growth by the MTT assay after 48 h drug exposure. g n.d means not determined. b c
Fig. 2. Cytotoxicity of cisplatin, carboplatin, oxaliplatin and complex 4 against seven human cancer cell lines by MTT assay.
35.92 18.27 5.43 10.93 1.26 25.50 27.40
Oxaliplatin
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Fig. 3. Cell cycle distribution of HCT-116 cells cultured in the absence or the presence of complex 4 at different concentrations.
HepG-2) for 24 h at a concentration of 50 mM, and the results are shown in Figs. 4 and 5. Four areas in the diagrams stand for unnatural death cells (positive for PI and negative for annexin/FITC, left square on the top), intact cells (negative for annexin and PI, left square at the bottom), death cells (positive for annexin and PI, right square on the top) and early apoptotic cells (negative for PI and positive for annexin, right square at the bottom), respectively. As shown in Fig. 6, complex 4, cisplatin and oxaliplatin greatly increased the apoptotic rate of the HCT-116 cells as compared with the untreated cells (control), suggesting that the tested compounds inhibited tumor cell growth by inducing apoptosis, especially for complex 4. As for HepG-2 cell line, the apoptotic rates induced by complex 4 and cisplatin were higher than that of oxaliplatin. Moreover, the apoptotic rates of complex 4 shown for HCT-116 and HepG-2 cell lines are 1.4-fold and 2.2-fold higher than those of cisplatin, which is consistent with the result of in vitro cytotoxicity assay, indicating that the anticancer efficacy of complex 4 is more potent than that of cisplatin. So it can be concluded that complex 4 can produce tumor cell death through an apoptotic pathway.
2.4. Interaction with pET28a plasmid DNA It is well known that DNA is the primary target for the platinumbased drugs. Thus, agarose gel electrophoresis was applied to study the interaction of pET28a plasmid DNA and complex 4, cisplatin and oxaliplatin. The tested compounds were incubated with DNA at different concentrations. In the electrophoretogram (Fig. 7), untreated pET28a plasmid DNA mainly consisted of covalently closed circular (Form I) and a small amount of nicked (Form II) bands. Owing to the unwinding of pET28a plasmid DNA, a decrease in the rate of migration for closed circular DNA (Form I) was observed for cisplatin and oxaliplatin. Moreover, a coalescence of the closed circular DNA (Form I) and open circular DNA (Form II) was observed for cisplatin, indicating a strong unwinding of the super coiled DNA. However, no migration was observed for complex 4, which is probably due to the fact that the large steric hindrance of complex 4 greatly reduces the interaction between the compound and DNA. Despite the kinetic reactivity of complex 4 with DNA is much slower than that of cisplatin, complex 4 still shows a little
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Fig. 4. Flow cytometric analysis of the distribution of HCT-116 cells treated with cisplatin oxaliplatin and complex 4 at 50 mM.
cytotoxicity superior to cisplatin against most tested tumor cell lines. This obviously hints that the interaction mode of complex 4 with DNA is different from that of cisplatin with DNA. Therefore, it may be reasonable to deduce that the strong sterically hindered effect has made complex 4 exert its cytotoxicity in a different way from cisplatin analogous complexes. 2.5. Western blot analysis To further explore the mechanism of action, three mitochondrial-related apoptotic proteins of Bax, Bcl-2 and Caspase3 were tested with HepG-2 and HCT-116 cells treated with cisplatin and complex 4, respectively, by western blot method. The results, as shown in Fig. 8, indicated that cisplatin and complex 4 could increase the ratio of Bax/Bcl-2 proteins both in HepG-2 cells and HCT116 cells. Besides, the downstream expression of prototype apoptotic executioner Caspase-3 was decreased and cleavedCaspase-3 was increased by cisplatin and complex 4, which could induce apoptosis of tumor cells. As we know, apoptosis is a highly complex and tightly regulated process involving many different signaling pathways. Among this, the platinum-induced apoptosis was to some extent caused by the mitochondrial-dependent apoptosis pathway.
3. Conclusion In the present investigation, a series of platinum(II) complexes of (1R,2R)-N1,N2-dibutyl-1,2-diaminocyclohexane as a carrier ligand with leaving groups like chloride and dicarboxylates, used in the current platinum based drugs, were designed and synthesized upon a concept that platinum complexes with certain steric hindrance in the Pt(II)-amine moiety could effectively improve the cytotoxicity and decrease the side effects. The in vitro cytotoxicity study revealed that complex 1 had negligible cytotoxicity against the tested cell lines, while complexes 2 and 3 showed selective activity against some tested cell lines. It is of significance to find that complex 4 showed extraordinary cytotoxicity against all the tested cancer cell lines, indicating that the function of alkyl moieties, as steric hindrance, on each amino group of 1,2-DACH has a significant influence on the antitumor property of the resulting platinum complexes in contrast to its parent compound, oxaliplatin. Definitely, the leaving group also plays an important role on the cytotoxicity of the newly synthesized platinum compounds. The flow cytometric analysis showed that the representative compound, complex 4, produced tumor cell death through an apoptotic pathway by blocking the cell cycle at G2/M phase. The agarose gel electrophoresis demonstrated that complex 4 has a distinct way
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Fig. 5. Flow cytometric analysis of the distribution of HepG-2 cells treated with cisplatin, oxaliplatin and complex 4 at 50 mM.
bound to DNA different from cisplatin, while Pt(II) complexes with N-monoalkyl 1R,2R-diaminocyclohexane derivatives as ligands showed a similar DNA binding pattern to that of cisplatin in our former reports. Furthermore, western blot analysis on complex 4 indicated it had a similar apoptotic mechanism to cisplatin which could induce apoptosis via a mitochondrial-dependent pathway. Consequently, the introduction of two alkyl moieties to the 1R,2RDACH skeleton as carrier ligand can lead to the formation of the corresponding platinum complexes with potent antitumor activity, and one of which can be a drug candidate for further study.
4. Experimental protocols 4.1. Chemistry 4.1.1. Materials and instruments All chemicals and solvents were of analytical reagent grade and were used without further purification. Potassium tetrachloroplatinate(II) was obtained from a local chemical company. The ligand was prepared by the method as described previously in our laboratory [29]. Silver dicarboxylates were prepared by the reaction of the corresponding sodium dicarboxylate with silver nitrate in water. Infrared spectra were measured on KBr pellets on a Nicolet IR200 FT-IR spectrometer in the range of 4000e400 cm1. 1 H NMR and 13C NMR spectra were recorded in DMSO with a Bruker 300 MHz NMR spectrometer. 195Pt spectra were recorded on a Bruker DRX500 spectrometer. Mass spectra were measured on an Agilent 6224 TOF LC/MS instrument. 4.1.2. Synthesis and characterization
Fig. 6. Flow cytometric analysis of the distribution of HepG-2 and HCT-116 cell lines untreated (control) or treated with cisplatin, oxaliplatin and complex 4 at 50 mM.
4.1.2.1. Complex 1. To an aqueous solution (20 mL) of K2PtCl4 (2.1 g, 5.0 mmoL) was added a solution of the ligand (1.1 g, 5.0 mmoL) in water (5 mL). The reaction mixture was heated to 60 C and stirred for 72 h in the dark. Plenty of yellow precipitate was filtered off, washed with water repeatedly, and then dried in vacuum.
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and 28.19 (CH2 of DACH), 27.40 (CH2CH2CH3), 45.32 (NHCH2), 64.15 (NHCH); 195Pt NMR (d6-DMSO, ppm): 2376.8; ESI-MS: m/z [MþNa]þ ¼ 515(100%), [2MþNa]þ ¼ 1007(60%). 4.1.2.4. Complex 2 (C16H30N2O4Pt). Yield: 15%, white powder. Elem anal. Calcd for C16H30N2O4Pt: C, 37.72; H, 5.93; N, 5.50. Found: C, 37.53; H, 5.68; N, 5.25. IR (KBr, cm1): 3128(br), 2937, 1698, 1672, 1596, 1395, 1310, 805, 775; 1H NMR (d6-DMSO/TMS, ppm): d 0.88e0.92 (m, 6H, CH2CH3), 0.93e1.19 (m, 16H, CH2 of DACH and CH3CH2CH2), 1.55e2.05 (m, 4H, NHCH2), 2.53e2.62 (m, 2H, NHCH), 6.11 (dd, 2H, CHNH; peaks disappeared after mixing with D2O); 13C NMR(d6-DMSO/TMS, ppm): d 13.84(CH3), 19.64(CH2CH3), 24.03 and 28.39(CH2 of DACH), 27.39(CH2CH2CH3), 46.98(NHCH2), 64.29(NHCH), 166(C]O); ESI-MS: m/z [MþH]þ ¼ 510(30%), [MþNa]þ ¼ 532(100%).
Fig. 7. Gel electrophoretic mobility pattern of pET28a plasmid DNA incubated with various concentrations of platinum(II) complexes. Lanes 1e8 (0, 10, 20, 40, 80, 160, 320, 640 mM) þ DNA. a) cisplatin; b) oxaliplatin; c) complex 4.
Fig. 8. HepG-2 and HCT-116 cells treated with 50 mM of cisplain and complex 4 for 12 h were examined for the expression of apoptosis-regulated proteins via using western blot analysis. Equal loading was testified by the detection of b-actin. Similar results were obtained from three independent experiments.
4.1.2.2. General synthesis of complexes 2e4. To a suspending aqueous solution (150 mL) containing 1 mmoL of complex 1, silver dicarboxylate (1mmoL) was added. The reaction mixture was heated to 40 C and stirred for 24 h under the lighting shielding condition. After the mixture was cooled to the room temperature, AgCl deposits were filtered off and washed with water. The filtrate was concentrated to 15 mL by a rotatory evaporator and then kept cool at 4 C for several hours. The resulting off-white solids were filtered off, washed with a small quantity of chilled water, and then dried in vacuum. 4.1.2.3. Complex 1 (C14H30Cl2N2Pt). Yield: 92%, yellow powder. Elem anal. Calcd for C14H30Cl2N2Pt: C, 34.15; H, 6.11; N, 5.70. Found: C, 34.33; H, 6.36; N, 5.48. IR (KBr, cm1): 3121(br), 2955, 2866, 1459, 1384, 1101, 973, 897, 778, 647; 1H NMR (d6-DMSO/TMS, ppm): d 0.88e0.98 (m, 6H, CH2CH3), 1.06e2.10 (m, 16H, CH2 of DACH and CH3CH2CH2), 2.26e2.41 (m, 4H, NHCH2), 3.03e3.11 (m, 2H, NHCH), 5.91 (dd, 2H, CHNH; peaks disappeared after mixing with D2O); 13C NMR(d6-DMSO/TMS, ppm): d 13.85 (CH3), 19.63 (CH2CH3), 24.29
4.1.2.5. Complex 3 (C17H32N2O4Pt). Yield: 22%, white powder. Elem anal. Calcd for C17H32N2O4Pt: C, 39.00; H, 6.12; N, 5.35. Found: C, 38.81; H, 6.01; N, 5.15. IR (KBr, cm1): 3501(br), 1645, 1571, 1435, 1356, 701; 1H NMR (d6-DMSO/TMS, ppm): d 0.90e1.11 (m, 6H, CH2CH3), 1.30e1.88 (m, 16H, CH2 of DACH and CH3CH2CH2), 2.36e2.53 (m, 4H, NHCH2), 2.75e2.86 (m, 2H, NHCH), 3.19e3.45 (s, 2H, (CO)2CH2), 5.45e6.10 (dd, 2H, CHNH; peaks disappeared after mixing with D2O); 13C NMR(d6-DMSO/TMS, ppm): 13.87 (CH3), 19.67 (CH2CH3), 24.09 and 28.32 (CH2 of DACH), 27.32 (CH2CH2CH3), 45.21 (NHCH2), 49.88 (CH2COO), 64.04 (NHCH), 173.40 (C]O); ESI-MS: m/z [MþNa]þ ¼ 546(100%). 4.1.2.6. Complex 4 (C20H36N2O4Pt). Yield: 35%, white powder. Elem anal. Calcd for C20H36N2O4Pt: C, 42.63; H, 6.39; N, 4.97. Found: C, 42.75; H, 6.47; N, 4.72. IR (KBr, cm1): 3429(br), 2944, 1596, 1410, 1333, 878; 1H NMR (d6-DMSO/TMS, ppm): d 0.90e0.93 (m, 6H, CH2CH3), 1.07e1.98 (m, 16H, CH2 of DACH and CH3CH2CH2), 2.20e2.39 (m, 6H, CH2CH2CH2), 2.53e2.74 (m, 4H, NHCH2), 2.75e3.17 (m, 2H, NHCH), 6.27 (dd, 2H, CHNH; peaks disappeared after mixing with D2O); 13C NMR (d6-DMSO/TMS, ppm): d 13.88 (CH3), 15.90 (CH2CH2CH2), 29.8 (CCH2CH2), 19.67(CH2CH3), 24.09 (CH2 of DACH), 28.32 (CH2 of DACH), 27.32 (CH2CH2CH3), 46.89 (NHCH2), 63.23 (COOeCCH2), 64.09 (NHCH), 177.7(C]O); 195Pt NMR (d6-DMSO, ppm): 2038.8; ESI-MS: m/z [MþNa]þ ¼ 586(55%), [2MþNa]þ ¼ 1149(100%). 4.2. Biological studies 4.2.1. In vitro cytotoxic activity In vitro cytotoxicity of the platinum compounds against human hepatocellular carcinoma (HepG-2), gastric carcinoma (SGC-7901), non-small-cell lung cancer (A549), and colorectal cancer (HCT-116) cell lines were measured by the MTT assays. Complex 4 was selected to measure the cytotoxicity against human breast carcinoma (MCF-7, MDA-MB-231), gastric carcinoma (BGC-823), leukemia (K562, NB4), prostatic cancer (DU145) and ovarian cancer (A2780) cell lines, respectively, by the same method. Briefly, the cells were seeded in 96-well cultured plates at a density of 5000 cells/well. After overnight incubation (16 h), the cells were treated with the platinum complexes. After 48 h of incubation, 10 mL of a freshly diluted 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2Htetrazolium bromide (MTT) solution (5 mg/mL) were added to each well and the plates were incubated at 37 C in a humidified 5% CO2 atmosphere for 4 h. At the end of the incubation period the medium was removed and the formazan product was dissolved in 150 mL of DMSO. The cell viability was evaluated by measurement of the absorbance at 490 nm, using an Absorbance Reader (BioRad). IC50 values (compound concentration that produces 50% of cell growth inhibition) were calculated from curves constructed by plotting cell
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survival inhibitory rate (%) versus drug concentration logarithm. All experiments were repeated in three times. The reading values were converted to the percentage of control (% cell survival). Cytotoxic effects were expressed as IC50 values. 4.2.2. Cell cycle measurement For the cell cycle study, 5 105 HCT-116 cells were seeded in 6well plate and incubated at 37 C in 5% CO2 overnight. The next day cells were treated with the platinum drugs for 24 h at the dose of 50 mM. Then cells were harvested with trypsin and washed twice with PBS. After that, cells were fixed in cold 70% ethanol and stored at 4 C for 12 h. On the day of analysis, ethanol was removed by centrifugation and cells were washed twice with PBS, then treated with RNase (75 kU/mL) for 30 min at 37 C. Propidium iodide (PI) was finally added (50 mg/mL) to stain the cellular DNA, and samples were processed by flow cytometer (FAC Scan, Becton Dickenson, USA). The 1 104 cells were acquired for each sample using the CELLQuest software and recording propidium iodide (PI) in FL2 channel. The cell cycle analysis was performed with ModFit software.
by BCA (bicinchoninic acid) protein assay (Thermo, Waltham, MA) and adjusted to the equal concentration. The samples (20 mg/lane) were separated in 8% or 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDSePAGE), and then transferred onto polyvinylidene difluoride (PVDF) Immobilon-P membrane (Bio-Rad, USA) by a transblot apparatus (Bio-Rad, USA). The membrane was blocked with 5% nonfat milk in TBST buffer for 1 h, followed by overnight incubation at 4 C with primary antibodies diluted in PBST (1:2000 b-actin, Santa Cruz, USA; 1:500 for Bax, BD Pharmagin, USA; 1:500 for Bcl-2, Cell Signal, USA). After washing with PBST, the membranes were incubated for 1 h with an IRDye™ 800 conjugated secondary antibody diluted 1:30,000 in PBST, and the labeled proteins were tested by an Odyssey Scanning System (LiCOR., Lincoln, Nebraska, USA). 4.2.6. Statistical analysis All the values of biological evaluation are the means ± SD of not less than three measurements. Statistical analysis was carried out using the Student's test. Acknowledgment
4.2.3. Induction of cell apoptosis HepG-2 and HCT-116 cell lines were grown in culture as description [22]. Cell apoptosis assays were performed as follows. In brief, cells were washed with PBS and digested by trypsin solution. A cell suspension was made with culture medium, and the concentration was adjusted to 3 105 cells/mL. Cells were plated into 6-well culture plates (2 mL/well) and incubated at 37 C in 5% CO2 overnight. A series of indicated doses of complexes were added into each well and incubated with cells for 24 h at 37 C in 5% CO2. Untreated cells were used as negative controls; Cisplatin was used as positive control. The apoptosis of cells was measured by Flow Cytometry using annexin V-FITC/PI apoptosis kit (Biouniquer, Nanjing, China) according to the manufacturer's instructions. Cells were harvested and washed in cold PBS, then stained with annexin V-FITC (100 ng/mL) and propidium iodide (2 mg/mL) in annexinbinding buffer (10 mM HEPES, 140 mM NaCl, 2.5 mM CaCl2, pH 7.4). After 15 min incubation at room temperature, the fluorescence of cells was measured using the flow cytometer (FACScan, Becton Dickenson, USA). The results were analyzed using CELLQuest software and represented as percentage of normal and apoptotic cells at various stages. FITC and PI fluorescence was measured in FL1 and FL2 channels, respectively. 4.2.4. Interaction with plasmid DNA Interaction of the platinum complexes with pET28a plasmid DNA was studied by agarose gel electrophoresis. Cisplatin and oxaliplatin were used as controls. In short, super coiled pET28a plasmid DNA (at concentration 0.5 mg/mL) were incubated with different platinum complexes at different dosages in HEPES buffer (25 mM HEPES, pH 7.4, 5 mM NaCl) in a water bath at 37 C in the dark for 20 h for DNA unwinding, respectively. Then 10 mL aliquots of drug-DNA mixtures were loaded onto the 1.0% agarose gel stained with GoldenView (Dingguo, China) and electrophoresis was carried under TAE buffer (0.05 M Tris base, 0.05 M glacial aceticacid, 1 mM EDTA, pH 7.5) for 60 min at 90 V. In the end of electrophoresis, the gels were then photographed under UV light (Gel Doc XR Imaging System, Bio-Rad). 4.2.5. Western blot analysis HepG-2 and HCT-116 cells were seeded in their respective culture vessel and incubated until 80% confluent. The cells were treated with platinum complexes at the concentration of 50 mmol for 12 h. The total cellular proteins were extracted by incubating the cells in the lysis buffer. The protein concentration was determined
We are grateful to the National Natural Science Foundation of China (Project 21272041) and the New Drug Creation Project of the National Science and Technology Major Foundation of China (Project 2013ZX09402102-001-006) as well as the Fundamental Research Funds for the Central Universities (Project 2242013K30011) for financial aids to this work. Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.ejmech.2015.04.019. References [1] B. Rosenberg, L. Vancamp, Nature 222 (1969) 385e386. [2] K.B. Huang, Z.F. Chen, Y.C. Liu, Z.Q. Li, J.H. Wei, W. Wang, H. Liang, Eur. J. Med. Chem. 64 (2013) 554e561. ^teaux, K. Brasseur, S. Fortin, V. Leblanc, S.P. Asselin, G. Be rube , [3] P. Saha, C. Desco Eur. J. Med. Chem. 48 (2012) 385e390. [4] V. Hristo, S.M. Valiahdi, A.A. Legin, M.A. Jakupec, A. Roller, M. Galanski, K.K. Keppler, Eur. J. Med. Chem. 46 (2011) 5456e5464. [5] J. Reedijk, Chem. Rev. 99 (1999) 2499e2510. [6] M.A. Jakupec, M. Galanski, B.K. Keppler, Rev. Physiol. Biochem. Pharmacol. 146 (2003) 1e53. [7] T. Soldatovic, Z.D. Bugarcic, J. Inorg. Biochem. 99 (2005) 1472e1479. [8] J. Zhao, S.H. Gou, G. Xu, L. Chen, Eur. J. Med. Chem. 85 (2014) 408e417. [9] A.K. Godwin, A. Meister, P.J. Dwyer, C.S. Huang, T.C. Hamilton, M.E. Anderson, Proc. Natl. Acad. Sci. 89 (1992) 3070e3074. [10] J.M. Woynarowski, W.G. Chapman, C. Napier, M.C.S. Herzig, P. Juniewicz, Mol. Pharmacol. 54 (1998) 770e777. [11] R.C. Todd, S.J. Lippard, Metallomics 1 (2009) 280e291. [12] B. Spingler, D.A. Whittington, S.J. Lippard, Inorg. Chem. 40 (2001) 5596e5602. [13] M. Galanski, A. Yasemi, M.A. Jakupec, N.G. Keyserlingk, B.K. Keppler, Monatsh. Chem. 136 (2005) 693e700. [14] S.A. Abramkin, U. Jungwirth, S.M. Valiahdi, C. Dworak, L. Habala, K. Meelich, W. Berger, M.A. Jakupec, C.G. Hartinger, A.A. Nazarov, B.K. Keppler, J. Med. Chem. 53 (2010) 7356e7364. [15] L. Habala, M. Galanski, A. Yasemi, A.A. Nazarov, N.G. Keyserlingk, B.K. Keppler, Eur. J. Med. Chem. 40 (2005) 1149e1155. [16] J. Gao, Y. Liu, Y. Zhou, R.A. Zingaro, ChemMedChem 2 (2007) 1723e1729. [17] J. Gao, Y. Liu, R.A. Zingaro, Chem. Res. Toxicol. 22 (2009) 1705e1712. [18] D.M. Fisher, P.J. Bednarski, R. Grünert, P. Turner, R.R. Fenton, J.R. AldrichWright, ChemMedChem 2 (2007) 488e495. ~ a, J.M. Mesas, M.-T. Alegre, M.C. Rodríguez, [19] J. Lorenzo, A. Delgado, A.M. Montan s, Eur. J. Med. Chem. 83 (2014) 374e388. F.-X. Avile rez, C.V. D-García, A. A-Lo pez, V. Solar, S. Cabrera, M.T. A-Ortun ~ o, C. N[20] C. Pe n, J.A. L-Martín, Eur. J. Med. Chem. 76 (2014) 360e368. Ranninger, J. Alema [21] A. Savi c, L. Filipovi c, S. AranCelovi c, B. Doj cinovi c, S. Radulovi c, T.J. Sabo, S. G Sipka, Eur. J. Med. Chem. 82 (2014) 372e384. [22] Y. Chen, J.A. Parkinson, Z. Guo, T. Brown, P.J. Sadler, Angew. Chem. Int. Ed. 38 (1999) 2060e2063. [23] F. Kanzawa, Y. Akiyama, N. Saijo, K. Nishio, Lung Cancer 40 (2003) 325e332.
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Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Cytotoxicity profile of novel sterically hindered platinum(II) complexes with (1R,2R)-N1,N2-dibutyl-1,2-diaminocyclohexane Haiyan Zhang a, Shaohua Gou a, b, *, Jian Zhao a, Feihong Chen a, b, Gang Xu a, b, Xia Liu c, ** a
Pharmaceutical Research Center and School of Chemistry and Chemical Engineering, Southeast University, Nanjing 211189, China Jiangsu Province Hi-Tech Key Laboratory for Bio-medical Research, Southeast University, Nanjing 211189, China c Department of Science and Technology, Jiangsu Open University, Nanjing 210036, China b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 7 November 2014 Received in revised form 6 April 2015 Accepted 7 April 2015 Available online 8 April 2015
Four Pt(II) complexes of (1R,2R)-N1,N2-dibutyl-1,2-diaminocyclohexane with two alkyl branches as steric hindrance have been designed and synthesized. In vitro cytotoxicity of these compounds indicated complex 4 is a cytotoxic agent more potent than its parent molecule, oxaliplatin, against almost all the tested cell lines. Agarose gel electrophoresis study showed that the kinetic reactivity of complex 4 with DNA is slow down due to the sterically hindered effect, demonstrating that it may possess a different mechanism of action from cisplatin. Flow cytometry results revealed that complex 4 induced apoptosis of tumor cells by blocking the cell-cycle progression in the G2/M phase. Western blot analysis showed it had a similar apoptotic mechanism to cisplatin which could induce apoptosis via a mitochondrialdependent pathway. © 2015 Elsevier Masson SAS. All rights reserved.
Keywords: Platinum (II) complexes Steric hindrance Potent cytotoxicity Apoptotic mechanism
1. Introduction Cisplatin and its analogs are one of the most important classes of chemotherapeutic agents available for the treatment of solid tumors [1e4]. However, the severe toxic side-effects and insurmountable cross-resistance are the major drawbacks of the present platinum-based drugs. It has been demonstrated that sulfurcontaining biomolecules such as glutathione and metallothionein have a high affinity for PtII complexes [5e8], which are responsible for the side effect and cross-resistance of the platinum(II) complexes. Moreover, the interaction of platinum(II) complexes with sulfur-containing molecules may deactivate the anticancer PtII complexes before the platinum complexes bind to DNA and form effective intrastrand Pt-DNA adducts [9]. Oxaliplatin as one of the best sale platinum based antitumor drugs has been found not to be affected in some cisplatin- and carboplatin-resistance tumors [10e12], this is believed to be largely attributed to its carrier ligand 1R,2R-diaminocyclohexane (1R,2R-DACH) which plays an important role in improving the characteristics of oxaliplatin by increasing lipophilicity and DNA mismatch repair inhibition [11,12].
* Corresponding author. Pharmaceutical Research Center and School of Chemistry and Chemical Engineering, Southeast University, Nanjing 211189, China. ** Corresponding author. E-mail addresses: [email protected], [email protected] (S. Gou). http://dx.doi.org/10.1016/j.ejmech.2015.04.019 0223-5234/© 2015 Elsevier Masson SAS. All rights reserved.
Encouraged by the successful application of 1R,2R-DACH in oxaliplatin, numerous 1,2-DACH derivatives have been designed and introduced to platinum complexes for antitumor study [13e18]. Among them, a small number of symmetric N1,N2-disubstituted 1,2-DACH derivatives have been used as carrier ligands for the improvement of the pharmacological profile of the present platinum based drugs [16e18]. But those ligands reported were not particular designed for increasing the steric hindrance of the resulting platinum(II) complexes to overcome the cross-resistance of the platinum(II) drugs. So far, much effort has been made to increase the steric hindrance of antitumor platinum(II) complexes [19e21], and some of approaches seem to have achieved the ideal results. For example, ZD0473 was synthesized by the introduction of 2-methylpyridine as a carrier ligand, which enables the platinum(II) complex to show considerable cytotoxicity against cancer cell lines, especially against cisplatin-resistant cancer cells with elevated glutathione and/or metallothionein levels due to the steric hindrance of 2methylpyridine [22,23]. Recently, our group has designed a series of platinum-based complexes bearing N-monosubstituted 1R,2Rdiaminocycloohexane derivatives as carrier ligands, aiming to enhance the sterically hindered effect of the resulting complexes [24,25]. The results indicated that N-monoalkyl derivatives of 1R,2R-DACH could effectively improve the pharmaceutical properties of the resulting platinum compounds as compared with
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oxaliplatin [26e28]. According to our previous study, we assumed that the steric hindrance of the substituted group may change the interaction mode between the platinum complexes and DNA, which may subsequently affect antitumor properties of the resulting complexes, thus leading to the absence of the cross resistance with cisplatin or oxaliplatin [27]. It is expected that the increased steric hindrance of platinum complexes can decrease the interaction between platinum complexes and sulfur-containing molecules and prolong time of blood cycle, resulting in enhancing curative effect for tumor. However, it should be kept in mind that huge steric hindrance with the designed platinum complexes may disturb and even prevent the interaction of the compound with DNA. As we believe that the moderate increase of sterically hindered effect of the platinum complexes can improve the cytotoxic activity and decrease the side effects of the present platinum-based drugs, we intend to introduce two alkyl moieties to the 1R,2R-DACH skeleton so as to obtain the leading platinum complexes which can take advantage of the successful leaving ligands like chloride ion in cisplatin, 1,1-cyclobutanedicarboxylate in carboplatin, malonate in heptaplatin and oxalate in oxaliplatin. Herein reported are four novel platinum complexes of (1R,2R)-N1,N2-dibutyl-1,2-DACH as a carrier ligands (Fig. 1) with their antitumor activity as well as tentative study on their mechanism of action.
2. Results and discussion 2.1. Chemistry Platinum(II) complexes were simply prepared in one or two steps following the procedures shown in Scheme 1. The interaction of the ligand (L) with potassium tetrachloroplatinate(II) led to the generation of complex 1. The reaction took a long time than expected, indicating that alkyl species have caused hindrance for the ligand to bind the metal atom. Further reaction of complex 1 with the corresponding silver dicarboxylate in water formed (1R,2R)N1,N2-dibutyl-1,2-DACH -Pt(II) conjugates (complexes 2e4), respectively. The newly synthesized platinum(II) complexes were characterized by microanalysis, IR, 1H and 13C NMR spectra with electrospray ionization mass (ESI-MS) spectroscopy. Complexes 1 and 4 were specially characterized by 195Pt NMR spectroscopy. In the infrared spectra, the NeH stretching vibrations of complexes 1e4 were obviously shifted to lower frequencies than those of free ligand [29], due to the amino group coordination with Pt(II) ions. Besides, the C]O vibration of complexes 2e4 appeared in the range from 1596 to 1698 cm1, characteristic of coordinated dicarboxylates, while the CeO feature appeared in the range of 1310e1356 cm1.
All the platinum(II) complexes show 100% of [MþNaþ] or [MþHþ] peaks, remarkably acompanied with three isotopes of platinum element, 194Pt (33%), 195Pt (34%), and 196Pt (25%), in their positive ESI-MS spectra. In the 1H NMR spectra, the signals of CH2 and CH protons next to the amino groups occurred in the range of 1.55e2.74 ppm and 2.53e3.17 ppm as multiplets, shifting to highfield relative to the corresponding signals (3.64e3.73 and 3.55e3.57 ppm) of the free ligand [29]. The broad signals of hydrogen atoms belonging to amino groups appeared in the range of 5.45e6.27 ppm due to the coordination with the metal atom, shifting to high-field compared with the metal-free ligand [29]. In the 195Pt spectra, both complexes 1 and 4 showed a single peak around 2000 ppm. Furthermore, the chemical shifts of 1H NMR and the numbers of carbon atoms of 13C NMR spectra for complexes 1e4 were in conformity with the proposed molecular structure of the compounds.
2.2. In vitro cytotoxic activity The cytotoxicity of the synthesized complexes was evaluated by MTT assays against human hepatocellular carcinoma cell line (HepG-2), gastric carcinoma cell line (SGC-7901), non-small-cell lung cancer cell line (A549), and colorectal cancer cell line (HCT116). Cisplatin and oxaliplatin were used as positive controls. The corresponding IC50 values are presented in Table 1. It is of much surprise to note that complex 1 had negligible cytotoxicity against all the tested cell lines, although chloride anions seemed to be easily separated from the Pt(II) ion as a leaving ligand in comparison to those dicarboxylates that served as bidentate ligands to bind the metal atom by chelation. Interestingly, both complexes 2 and 3 showed selective activity against certain tested cell lines. The former was sensitive to A549 and HCT-116 cell lines with IC50 values comparable to cisplatin and oxaliplatin, while the latter only expressed activity toward HepG-2 cell line with an IC50 value junior to the positive controls. However, complex 4 showed extraordinary cytotoxicity against all the test cell lines, which notably showed high cytotoxicity superior to that of cisplatin or oxaliplatin against HepG-2, A549 and HCT-116 cell lines except SGC-7901. Especially, complex 4 was 2.6-fold as potent as oxaliplatin toward human colorectal cancer cell line (HCT-116) that is the most sensitive to oxaliplatin among the present platinum based drugs. By comparing the cytotoxicity of complexes 1e4 and positive controls, it is rational to conclude that the existence of alkyl moieties, as steric hindrance, on each amino group of 1,2-DACH has a significant influence on the antitumor property of the resulting platinum complexes in contrast to its parent compound, oxaliplatin. Nevertheless, the role of the leaving ligands on the cytotoxicity of
Fig. 1. Related antitumor Pt(II) complexes in this paper.
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Scheme 1. Preparation of complexes 1e4.
moieties to the 1R,2R-DACH skeleton is an effective way to improve the antitumor activity of the resulting platinum(II) complex by increasing the steric hindrance in complex 4.
Table 1 In vitro cytotoxicity of complexes 1e4, cisplatin and oxaliplatin. Complexes
1 2 3 4 Cisplatin Oxaliplatin
IC50 (mM)a HepG-2
SGC-7901
A549
HCT-116
>100 >100 20.3 ± 1.56 6.5 ± 0.55 7.2 ± 0.84 6.6 ± 0.56
>100 >100 >100 16.9 ± 1.58 2.5 ± 0.23 4.2 ± 0.35
>100 28.3 ± 2.34 >100 8.5 ± 0.76 13.3 ± 0.12 12.5 ± 1.13
>100 30.1 ± 2.85 >100 7.3 ± 0.66 29.3 ± 0.24 19.1 ± 1.89
a Mean value ± standard deviation from three independent experiments; IC50 defined as the drug concentration required to inhibit 50% of cell growth by the MTT assay after 48 h drug exposure.
the newly synthesized platinum compounds can not be ignored. It appears that 1,1-cyclobutanedicarboxylate is a suitable leaving group for the platinum complex with (1R,2R)-N1,N2-dibutyl-1,2diaminocyclohexane as a carrier ligand. Considering complex 4 with much potent cytotoxicity in the preliminary assays, we further investigated its cytotoxicity against the other human cancer cell lines, including breast carcinoma cell lines (MCF-7, MDA-MB-231), gastric carcinoma cell line (BGC-823), leukemia cell lines (K562, NB4), prostatic cancer cell line (DU145), and ovarian cancer cell line (A2780) (Fig. 2). The corresponding IC50 data and those of cisplatin, oxaliplatin and carboplatin as positive controls are listed in Table 2. As expected, complex 4 showed potent cytotoxicity against all the tested cell lines. Notably, it even exhibited superior cytotoxicity to cisplatin in MCF-7, MDA-MB-231, K562, DU145 and A2780 cell lines. In addition, complex 4 was more potent than its parent molecule, oxaliplatin, against almost all the tested cell lines, indicating that it is a potent cytotoxic agent that possesses a broad antitumor spectrum of activity. It is of much interest to note that carboplatin and complex 4 have the same leaving group of 1,1-cyclobutanedicarboxylate, but the biological activity of carboplatin is much weaker than that of our compound. Again, the results indicated that the introduction of two alkyl
2.3. Flow cytometry study 2.3.1. Cell cycle analysis It is generally accepted that the cell cycle arresting induced by a platinum compound is necessary because it enables the nucleotide excision repair (NER) complex to remove the adducts and promote cell survival. When repair is incomplete, the cells will die by apoptosis. For instance, cisplatin inhibits cancer cell growth by blocking the cell cycle through the S or G2/M phase [30,31]. In order to preliminarily reveal the mechanism of action of the synthesized complexes, flow cytometric analysis was applied to detect the inhibitory effect of complex 4 on HCT-116 cell cycle progression. The results are shown in Fig. 3. Obviously, this compound induced S phase cell depletion, suggesting that apoptosis was launched from the S phase, and an accumulation of cells in the G2/M phase was observed as compared with the control tumor cells, which is in accordance with the effect of most anticancer agents. Moreover, the percentage of G2/M phase increased with the concentration of complex 4 increasing from 5 to 20 mM. In general, the compound appeared to induce apoptosis in a dose-dependent manner in HCT-116 cells by the increase of cell population at G2/ M phase. 2.3.2. Apoptosis analysis With the purpose of detecting in which way complex 4 induced cellular death (necrosis or apoptosis), the apoptotic analysis of complex 4, cisplatin and oxaliplatin against HCT-116 and HepG-2 cells was performed by using an Annexin V-FITC/PI assay. The tested compounds were incubated with tumor cells (HCT-116 and
Table 2 Further in vitro assay of complex 4 with cisplatin, oxaliplatin, carboplatin as positive controls. Cell lines
IC50 (mM)f Cisplatin
a
MCF-7 MDA-MB-231a BGC-823b K562c NB4c DU145d A2780e a
± ± ± ± ± ± ±
3.56 1.72 0.51 1.08 0.13 2.35 1.54
25.71 26.82 7.91 39.92 52.82 20.40 6.58
± ± ± ± ± ± ±
1.67 2.25 0.65 2.65 3.76 1.86 0.69
Carboplatin
Complex 4
109.47 ± 8.76 112.26 ± 8.76 43.21 ± 2.54 n.d. 12.59 ± 2.43 n.d.g 17.20 ± 2.41
17.52 13.79 8.28 4.51 9.68 5.05 4.55
± ± ± ± ± ± ±
1.67 5.89 0.74 0.32 0.15 0.43 0.33
Human breast carcinoma cell lines. Human gastric carcinoma cell line. Leukemia cell lines(K562,NB4). d Human prostatic cancer cell line. e Human ovarian cancer cell line. f Mean value ± Standard Deviation from three independent experiments; IC50 defined as the drug concentration required to inhibit 50% of cell growth by the MTT assay after 48 h drug exposure. g n.d means not determined. b c
Fig. 2. Cytotoxicity of cisplatin, carboplatin, oxaliplatin and complex 4 against seven human cancer cell lines by MTT assay.
35.92 18.27 5.43 10.93 1.26 25.50 27.40
Oxaliplatin
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Fig. 3. Cell cycle distribution of HCT-116 cells cultured in the absence or the presence of complex 4 at different concentrations.
HepG-2) for 24 h at a concentration of 50 mM, and the results are shown in Figs. 4 and 5. Four areas in the diagrams stand for unnatural death cells (positive for PI and negative for annexin/FITC, left square on the top), intact cells (negative for annexin and PI, left square at the bottom), death cells (positive for annexin and PI, right square on the top) and early apoptotic cells (negative for PI and positive for annexin, right square at the bottom), respectively. As shown in Fig. 6, complex 4, cisplatin and oxaliplatin greatly increased the apoptotic rate of the HCT-116 cells as compared with the untreated cells (control), suggesting that the tested compounds inhibited tumor cell growth by inducing apoptosis, especially for complex 4. As for HepG-2 cell line, the apoptotic rates induced by complex 4 and cisplatin were higher than that of oxaliplatin. Moreover, the apoptotic rates of complex 4 shown for HCT-116 and HepG-2 cell lines are 1.4-fold and 2.2-fold higher than those of cisplatin, which is consistent with the result of in vitro cytotoxicity assay, indicating that the anticancer efficacy of complex 4 is more potent than that of cisplatin. So it can be concluded that complex 4 can produce tumor cell death through an apoptotic pathway.
2.4. Interaction with pET28a plasmid DNA It is well known that DNA is the primary target for the platinumbased drugs. Thus, agarose gel electrophoresis was applied to study the interaction of pET28a plasmid DNA and complex 4, cisplatin and oxaliplatin. The tested compounds were incubated with DNA at different concentrations. In the electrophoretogram (Fig. 7), untreated pET28a plasmid DNA mainly consisted of covalently closed circular (Form I) and a small amount of nicked (Form II) bands. Owing to the unwinding of pET28a plasmid DNA, a decrease in the rate of migration for closed circular DNA (Form I) was observed for cisplatin and oxaliplatin. Moreover, a coalescence of the closed circular DNA (Form I) and open circular DNA (Form II) was observed for cisplatin, indicating a strong unwinding of the super coiled DNA. However, no migration was observed for complex 4, which is probably due to the fact that the large steric hindrance of complex 4 greatly reduces the interaction between the compound and DNA. Despite the kinetic reactivity of complex 4 with DNA is much slower than that of cisplatin, complex 4 still shows a little
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Fig. 4. Flow cytometric analysis of the distribution of HCT-116 cells treated with cisplatin oxaliplatin and complex 4 at 50 mM.
cytotoxicity superior to cisplatin against most tested tumor cell lines. This obviously hints that the interaction mode of complex 4 with DNA is different from that of cisplatin with DNA. Therefore, it may be reasonable to deduce that the strong sterically hindered effect has made complex 4 exert its cytotoxicity in a different way from cisplatin analogous complexes. 2.5. Western blot analysis To further explore the mechanism of action, three mitochondrial-related apoptotic proteins of Bax, Bcl-2 and Caspase3 were tested with HepG-2 and HCT-116 cells treated with cisplatin and complex 4, respectively, by western blot method. The results, as shown in Fig. 8, indicated that cisplatin and complex 4 could increase the ratio of Bax/Bcl-2 proteins both in HepG-2 cells and HCT116 cells. Besides, the downstream expression of prototype apoptotic executioner Caspase-3 was decreased and cleavedCaspase-3 was increased by cisplatin and complex 4, which could induce apoptosis of tumor cells. As we know, apoptosis is a highly complex and tightly regulated process involving many different signaling pathways. Among this, the platinum-induced apoptosis was to some extent caused by the mitochondrial-dependent apoptosis pathway.
3. Conclusion In the present investigation, a series of platinum(II) complexes of (1R,2R)-N1,N2-dibutyl-1,2-diaminocyclohexane as a carrier ligand with leaving groups like chloride and dicarboxylates, used in the current platinum based drugs, were designed and synthesized upon a concept that platinum complexes with certain steric hindrance in the Pt(II)-amine moiety could effectively improve the cytotoxicity and decrease the side effects. The in vitro cytotoxicity study revealed that complex 1 had negligible cytotoxicity against the tested cell lines, while complexes 2 and 3 showed selective activity against some tested cell lines. It is of significance to find that complex 4 showed extraordinary cytotoxicity against all the tested cancer cell lines, indicating that the function of alkyl moieties, as steric hindrance, on each amino group of 1,2-DACH has a significant influence on the antitumor property of the resulting platinum complexes in contrast to its parent compound, oxaliplatin. Definitely, the leaving group also plays an important role on the cytotoxicity of the newly synthesized platinum compounds. The flow cytometric analysis showed that the representative compound, complex 4, produced tumor cell death through an apoptotic pathway by blocking the cell cycle at G2/M phase. The agarose gel electrophoresis demonstrated that complex 4 has a distinct way
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Fig. 5. Flow cytometric analysis of the distribution of HepG-2 cells treated with cisplatin, oxaliplatin and complex 4 at 50 mM.
bound to DNA different from cisplatin, while Pt(II) complexes with N-monoalkyl 1R,2R-diaminocyclohexane derivatives as ligands showed a similar DNA binding pattern to that of cisplatin in our former reports. Furthermore, western blot analysis on complex 4 indicated it had a similar apoptotic mechanism to cisplatin which could induce apoptosis via a mitochondrial-dependent pathway. Consequently, the introduction of two alkyl moieties to the 1R,2RDACH skeleton as carrier ligand can lead to the formation of the corresponding platinum complexes with potent antitumor activity, and one of which can be a drug candidate for further study.
4. Experimental protocols 4.1. Chemistry 4.1.1. Materials and instruments All chemicals and solvents were of analytical reagent grade and were used without further purification. Potassium tetrachloroplatinate(II) was obtained from a local chemical company. The ligand was prepared by the method as described previously in our laboratory [29]. Silver dicarboxylates were prepared by the reaction of the corresponding sodium dicarboxylate with silver nitrate in water. Infrared spectra were measured on KBr pellets on a Nicolet IR200 FT-IR spectrometer in the range of 4000e400 cm1. 1 H NMR and 13C NMR spectra were recorded in DMSO with a Bruker 300 MHz NMR spectrometer. 195Pt spectra were recorded on a Bruker DRX500 spectrometer. Mass spectra were measured on an Agilent 6224 TOF LC/MS instrument. 4.1.2. Synthesis and characterization
Fig. 6. Flow cytometric analysis of the distribution of HepG-2 and HCT-116 cell lines untreated (control) or treated with cisplatin, oxaliplatin and complex 4 at 50 mM.
4.1.2.1. Complex 1. To an aqueous solution (20 mL) of K2PtCl4 (2.1 g, 5.0 mmoL) was added a solution of the ligand (1.1 g, 5.0 mmoL) in water (5 mL). The reaction mixture was heated to 60 C and stirred for 72 h in the dark. Plenty of yellow precipitate was filtered off, washed with water repeatedly, and then dried in vacuum.
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and 28.19 (CH2 of DACH), 27.40 (CH2CH2CH3), 45.32 (NHCH2), 64.15 (NHCH); 195Pt NMR (d6-DMSO, ppm): 2376.8; ESI-MS: m/z [MþNa]þ ¼ 515(100%), [2MþNa]þ ¼ 1007(60%). 4.1.2.4. Complex 2 (C16H30N2O4Pt). Yield: 15%, white powder. Elem anal. Calcd for C16H30N2O4Pt: C, 37.72; H, 5.93; N, 5.50. Found: C, 37.53; H, 5.68; N, 5.25. IR (KBr, cm1): 3128(br), 2937, 1698, 1672, 1596, 1395, 1310, 805, 775; 1H NMR (d6-DMSO/TMS, ppm): d 0.88e0.92 (m, 6H, CH2CH3), 0.93e1.19 (m, 16H, CH2 of DACH and CH3CH2CH2), 1.55e2.05 (m, 4H, NHCH2), 2.53e2.62 (m, 2H, NHCH), 6.11 (dd, 2H, CHNH; peaks disappeared after mixing with D2O); 13C NMR(d6-DMSO/TMS, ppm): d 13.84(CH3), 19.64(CH2CH3), 24.03 and 28.39(CH2 of DACH), 27.39(CH2CH2CH3), 46.98(NHCH2), 64.29(NHCH), 166(C]O); ESI-MS: m/z [MþH]þ ¼ 510(30%), [MþNa]þ ¼ 532(100%).
Fig. 7. Gel electrophoretic mobility pattern of pET28a plasmid DNA incubated with various concentrations of platinum(II) complexes. Lanes 1e8 (0, 10, 20, 40, 80, 160, 320, 640 mM) þ DNA. a) cisplatin; b) oxaliplatin; c) complex 4.
Fig. 8. HepG-2 and HCT-116 cells treated with 50 mM of cisplain and complex 4 for 12 h were examined for the expression of apoptosis-regulated proteins via using western blot analysis. Equal loading was testified by the detection of b-actin. Similar results were obtained from three independent experiments.
4.1.2.2. General synthesis of complexes 2e4. To a suspending aqueous solution (150 mL) containing 1 mmoL of complex 1, silver dicarboxylate (1mmoL) was added. The reaction mixture was heated to 40 C and stirred for 24 h under the lighting shielding condition. After the mixture was cooled to the room temperature, AgCl deposits were filtered off and washed with water. The filtrate was concentrated to 15 mL by a rotatory evaporator and then kept cool at 4 C for several hours. The resulting off-white solids were filtered off, washed with a small quantity of chilled water, and then dried in vacuum. 4.1.2.3. Complex 1 (C14H30Cl2N2Pt). Yield: 92%, yellow powder. Elem anal. Calcd for C14H30Cl2N2Pt: C, 34.15; H, 6.11; N, 5.70. Found: C, 34.33; H, 6.36; N, 5.48. IR (KBr, cm1): 3121(br), 2955, 2866, 1459, 1384, 1101, 973, 897, 778, 647; 1H NMR (d6-DMSO/TMS, ppm): d 0.88e0.98 (m, 6H, CH2CH3), 1.06e2.10 (m, 16H, CH2 of DACH and CH3CH2CH2), 2.26e2.41 (m, 4H, NHCH2), 3.03e3.11 (m, 2H, NHCH), 5.91 (dd, 2H, CHNH; peaks disappeared after mixing with D2O); 13C NMR(d6-DMSO/TMS, ppm): d 13.85 (CH3), 19.63 (CH2CH3), 24.29
4.1.2.5. Complex 3 (C17H32N2O4Pt). Yield: 22%, white powder. Elem anal. Calcd for C17H32N2O4Pt: C, 39.00; H, 6.12; N, 5.35. Found: C, 38.81; H, 6.01; N, 5.15. IR (KBr, cm1): 3501(br), 1645, 1571, 1435, 1356, 701; 1H NMR (d6-DMSO/TMS, ppm): d 0.90e1.11 (m, 6H, CH2CH3), 1.30e1.88 (m, 16H, CH2 of DACH and CH3CH2CH2), 2.36e2.53 (m, 4H, NHCH2), 2.75e2.86 (m, 2H, NHCH), 3.19e3.45 (s, 2H, (CO)2CH2), 5.45e6.10 (dd, 2H, CHNH; peaks disappeared after mixing with D2O); 13C NMR(d6-DMSO/TMS, ppm): 13.87 (CH3), 19.67 (CH2CH3), 24.09 and 28.32 (CH2 of DACH), 27.32 (CH2CH2CH3), 45.21 (NHCH2), 49.88 (CH2COO), 64.04 (NHCH), 173.40 (C]O); ESI-MS: m/z [MþNa]þ ¼ 546(100%). 4.1.2.6. Complex 4 (C20H36N2O4Pt). Yield: 35%, white powder. Elem anal. Calcd for C20H36N2O4Pt: C, 42.63; H, 6.39; N, 4.97. Found: C, 42.75; H, 6.47; N, 4.72. IR (KBr, cm1): 3429(br), 2944, 1596, 1410, 1333, 878; 1H NMR (d6-DMSO/TMS, ppm): d 0.90e0.93 (m, 6H, CH2CH3), 1.07e1.98 (m, 16H, CH2 of DACH and CH3CH2CH2), 2.20e2.39 (m, 6H, CH2CH2CH2), 2.53e2.74 (m, 4H, NHCH2), 2.75e3.17 (m, 2H, NHCH), 6.27 (dd, 2H, CHNH; peaks disappeared after mixing with D2O); 13C NMR (d6-DMSO/TMS, ppm): d 13.88 (CH3), 15.90 (CH2CH2CH2), 29.8 (CCH2CH2), 19.67(CH2CH3), 24.09 (CH2 of DACH), 28.32 (CH2 of DACH), 27.32 (CH2CH2CH3), 46.89 (NHCH2), 63.23 (COOeCCH2), 64.09 (NHCH), 177.7(C]O); 195Pt NMR (d6-DMSO, ppm): 2038.8; ESI-MS: m/z [MþNa]þ ¼ 586(55%), [2MþNa]þ ¼ 1149(100%). 4.2. Biological studies 4.2.1. In vitro cytotoxic activity In vitro cytotoxicity of the platinum compounds against human hepatocellular carcinoma (HepG-2), gastric carcinoma (SGC-7901), non-small-cell lung cancer (A549), and colorectal cancer (HCT-116) cell lines were measured by the MTT assays. Complex 4 was selected to measure the cytotoxicity against human breast carcinoma (MCF-7, MDA-MB-231), gastric carcinoma (BGC-823), leukemia (K562, NB4), prostatic cancer (DU145) and ovarian cancer (A2780) cell lines, respectively, by the same method. Briefly, the cells were seeded in 96-well cultured plates at a density of 5000 cells/well. After overnight incubation (16 h), the cells were treated with the platinum complexes. After 48 h of incubation, 10 mL of a freshly diluted 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2Htetrazolium bromide (MTT) solution (5 mg/mL) were added to each well and the plates were incubated at 37 C in a humidified 5% CO2 atmosphere for 4 h. At the end of the incubation period the medium was removed and the formazan product was dissolved in 150 mL of DMSO. The cell viability was evaluated by measurement of the absorbance at 490 nm, using an Absorbance Reader (BioRad). IC50 values (compound concentration that produces 50% of cell growth inhibition) were calculated from curves constructed by plotting cell
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survival inhibitory rate (%) versus drug concentration logarithm. All experiments were repeated in three times. The reading values were converted to the percentage of control (% cell survival). Cytotoxic effects were expressed as IC50 values. 4.2.2. Cell cycle measurement For the cell cycle study, 5 105 HCT-116 cells were seeded in 6well plate and incubated at 37 C in 5% CO2 overnight. The next day cells were treated with the platinum drugs for 24 h at the dose of 50 mM. Then cells were harvested with trypsin and washed twice with PBS. After that, cells were fixed in cold 70% ethanol and stored at 4 C for 12 h. On the day of analysis, ethanol was removed by centrifugation and cells were washed twice with PBS, then treated with RNase (75 kU/mL) for 30 min at 37 C. Propidium iodide (PI) was finally added (50 mg/mL) to stain the cellular DNA, and samples were processed by flow cytometer (FAC Scan, Becton Dickenson, USA). The 1 104 cells were acquired for each sample using the CELLQuest software and recording propidium iodide (PI) in FL2 channel. The cell cycle analysis was performed with ModFit software.
by BCA (bicinchoninic acid) protein assay (Thermo, Waltham, MA) and adjusted to the equal concentration. The samples (20 mg/lane) were separated in 8% or 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDSePAGE), and then transferred onto polyvinylidene difluoride (PVDF) Immobilon-P membrane (Bio-Rad, USA) by a transblot apparatus (Bio-Rad, USA). The membrane was blocked with 5% nonfat milk in TBST buffer for 1 h, followed by overnight incubation at 4 C with primary antibodies diluted in PBST (1:2000 b-actin, Santa Cruz, USA; 1:500 for Bax, BD Pharmagin, USA; 1:500 for Bcl-2, Cell Signal, USA). After washing with PBST, the membranes were incubated for 1 h with an IRDye™ 800 conjugated secondary antibody diluted 1:30,000 in PBST, and the labeled proteins were tested by an Odyssey Scanning System (LiCOR., Lincoln, Nebraska, USA). 4.2.6. Statistical analysis All the values of biological evaluation are the means ± SD of not less than three measurements. Statistical analysis was carried out using the Student's test. Acknowledgment
4.2.3. Induction of cell apoptosis HepG-2 and HCT-116 cell lines were grown in culture as description [22]. Cell apoptosis assays were performed as follows. In brief, cells were washed with PBS and digested by trypsin solution. A cell suspension was made with culture medium, and the concentration was adjusted to 3 105 cells/mL. Cells were plated into 6-well culture plates (2 mL/well) and incubated at 37 C in 5% CO2 overnight. A series of indicated doses of complexes were added into each well and incubated with cells for 24 h at 37 C in 5% CO2. Untreated cells were used as negative controls; Cisplatin was used as positive control. The apoptosis of cells was measured by Flow Cytometry using annexin V-FITC/PI apoptosis kit (Biouniquer, Nanjing, China) according to the manufacturer's instructions. Cells were harvested and washed in cold PBS, then stained with annexin V-FITC (100 ng/mL) and propidium iodide (2 mg/mL) in annexinbinding buffer (10 mM HEPES, 140 mM NaCl, 2.5 mM CaCl2, pH 7.4). After 15 min incubation at room temperature, the fluorescence of cells was measured using the flow cytometer (FACScan, Becton Dickenson, USA). The results were analyzed using CELLQuest software and represented as percentage of normal and apoptotic cells at various stages. FITC and PI fluorescence was measured in FL1 and FL2 channels, respectively. 4.2.4. Interaction with plasmid DNA Interaction of the platinum complexes with pET28a plasmid DNA was studied by agarose gel electrophoresis. Cisplatin and oxaliplatin were used as controls. In short, super coiled pET28a plasmid DNA (at concentration 0.5 mg/mL) were incubated with different platinum complexes at different dosages in HEPES buffer (25 mM HEPES, pH 7.4, 5 mM NaCl) in a water bath at 37 C in the dark for 20 h for DNA unwinding, respectively. Then 10 mL aliquots of drug-DNA mixtures were loaded onto the 1.0% agarose gel stained with GoldenView (Dingguo, China) and electrophoresis was carried under TAE buffer (0.05 M Tris base, 0.05 M glacial aceticacid, 1 mM EDTA, pH 7.5) for 60 min at 90 V. In the end of electrophoresis, the gels were then photographed under UV light (Gel Doc XR Imaging System, Bio-Rad). 4.2.5. Western blot analysis HepG-2 and HCT-116 cells were seeded in their respective culture vessel and incubated until 80% confluent. The cells were treated with platinum complexes at the concentration of 50 mmol for 12 h. The total cellular proteins were extracted by incubating the cells in the lysis buffer. The protein concentration was determined
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