Preclinical report 957
Trastuzumab-mediated selective delivery for platinum drug to HER2-positive breast cancer cells Rong Huanga, Yu Suna,b, Qihe Gaoc, Qiucui Wanga and Baiwang Suna Oxaliplatin is used widely as an anticancer drug for clinical treatment. However, its applications are limited because of its poor selectivity. In this work, we described the design, synthesis, and characterization of conjugates combining trastuzumab with a platinum (IV) analog of oxaliplatin, in which the trastuzumab acted as an active targeting agent for HER2-positive cancer cells. Indirect enzyme-linked immunosorbent assay and immunofluorescence study indicated the platinum (IV)–trastuzumab conjugates retained specific binding activity to HER2 overexpressed SK-BR-3 cells. In the presence of ascorbic acid, platinum (IV)–trastuzumab conjugates were reduced to platinum (II) analogs, which could bind to and unwind PUC19 DNA in a manner similar to oxaliplatin. The cytotoxic study was tested on three breast cell lines: SK-BR-3, MCF-7, and MDAMB-231. Platinum (IV)–trastuzumab conjugates showed promising antiproliferative activity against SK-BR-3 cells,
Introduction Platinum-based antitumor drugs are used widely for the treatment of various malignancies such as genitourinary, colorectal, and non-small-cell lung cancers [1,2]. Compared with cisplatin and carboplatin, oxaliplatin (Fig. 1) has a slightly different mode of action. The chelating oxalate ligand is more inert as a leaving group compared with chlorides, lowering the aquation rate and avoiding drug resistance [3]. Oxaliplatin is highly active in colorectal cancer and has shown very promising activity in metastatic breast cancer either in monotherapy or in combination with 5-fluorouracil or trastuzumab [4]. However, the application of oxaliplatin is limited by severe side effects, such as nephrotoxicity, ototoxicity, and neurotoxicity, and its poor ability to distinguish healthy and cancer cells. As a result, there has been considerable focus on the development of targeting delivery for platinum drugs. It is widely accepted that platinum (IV) compounds act as potential prodrugs, which are believed to be activated in the cancer cells by being reduced to platinum (II) analogs [5–7]. The platinum (IV) compounds, with an octahedral coordination structure, are kinetically inert compared with platinum (II) compounds [8]. The two extra ligands introduced into the platinum center can be modified for drug-targeting strategies [9–11], which are lost during Supplemental digital content is available for this article. Direct URL citations appear in the printed text and are provided in the HTML and PDF versions of this article on the journal's website (www.anti-cancerdrugs.com). 0959-4973 Copyright © 2015 Wolters Kluwer Health, Inc. All rights reserved.
but significantly decreased the inhibition to MDA-MB-231 and MCF-7 cells. The flow cytometric analysis showed that the conjugates arrested the cell cycle mainly at the G2/M phase and killed the cells through an apoptotic pathway. Anti-Cancer Drugs 26:957–963 Copyright © 2015 Wolters Kluwer Health, Inc. All rights reserved. Anti-Cancer Drugs 2015, 26:957–963 Keywords: antibody drug conjugate, anticancer drug, cell cycle arrest, platinum (IV) drug, targeting delivery a
School of Chemistry and Chemical Engineering, Southeast University, Nanjing, School of Pharmacy, Wannan Medical College, Wuhu and cInstitute of Microbiology, Chinese Academy of Science, Beijing, People’s Republic of China b
Correspondence to Baiwang Sun, School of Chemistry and Chemical Engineering, Southeast University, Nanjing 211189, People’s Republic of China Tel/fax: + 86 25 5209 0614; e-mail: [email protected] Received 26 March 2015 Revised form accepted 23 June 2015
reduction and are not involved in the mechanism of cell killing. Folic acid, estrogens, aptamers, and peptides have already been reported as targeting agents for platinum drugs delivery [12–15]. For the past few years, therapeutic antibodies have been considered promising targeting agents because of their low immunogenicity and high affinity to cancer cells [16,17]. Trastuzumab is a humanized monoclonal antibody that can specifically bind to the cell surface receptor, human epidermal growth factor receptor 2 (HER2). It has been considered a successful targeting agent for anticancer drugs delivery [18–20]. Thus, we chose trastuzumab as a representative antibody in our study. In this work, we designed a drug delivery strategy based on the synthesized platinum (IV) compound 2 (Fig. 1) using trastuzumab as an active cancer targeting agent. The conjugates are supposed to retain the specific binding ability to targeted cancer cells and confer additional cell-killing activity after reducing to platinum (II) analogs in cells. To test this assumption, three breast cell lines with different HER2 expression levels, SK-BR-3, MCF-7, and MDAMB-231, were selected. Indirect enzyme-linked immunosorbent assay (ELISA) assay, immunofluorescence study, and cytotoxicity study were carried out in this study to investigate the targeting binding activity and cytotoxicity of conjugates. Furthermore, gel electrophoresis and flow cytometry were used to discuss the DNA binding and cellkilling mode of conjugates. DOI: 10.1097/CAD.0000000000000272
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Anti-Cancer Drugs 2015, Vol 26 No 9
Fig. 1
O OH O
OH H2 N
O
O
H2 N
O
Pt N H2
H2 N
O
O
N H2
O
O
O
O
Pt
Pt O
O
O
O
O
N H2 O
OH HO Oxaliplatin
O
1
2
Structures of oxaliplatin, 1 and 2.
Materials and methods Materials
Oxaliplatin was obtained from Shangdong Boyuan Pharmaceutical Co. Ltd (Shangdong, China). Trastuzumab (10 mg/ml) was purchased from Harbin Pharmaceutical Group Bioengineering Co. Ltd (Harbin, China). All other chemicals were of analytical grade and used without further purification. HER2 protein was purchased from R&D Systems Inc. (Minneapolis, Minnesota, USA). Horseradish peroxidase-conjugated goat anti-human IgG (Fab)′, fluorescein isothiocyanate (FITC)-labeled goat anti-human IgG (Fab)′, and 4′,6-diamidino-2-phenylindole (DAPI) were purchased from Sigma-Aldrich (St. Louis, Missouri, USA). Cell counting kit-8 (CCK-8) was obtained from Dojindo Molecular Technologies (Kumamoto, Japan). 3,3′,5,5′Tetramethylbenzidine (TMB) solution was obtained from the Beyotime Institute of Biotechnology (Shanghai, China). The PUC19 plasmid DNA substrate was purchased from New England Biolabs (Ipswich, Massachusetts, USA) and propidium iodide (PI) was purchased from Becton Dickinson and Company (Franklin Lakes, New Jersey, USA). Preparation of trastuzumab–2 conjugates Synthesis of trans-R,R-1,2-diaminocyclohexane dihydroxo(oxalate)platinum (IV) (1)
Compound 1 was synthesized in a similar manner according to the literature procedures [21,22]. Briefly, hydrogen peroxide (30 wt%, 20 ml) was added dropwise to a suspension of oxaliplatin (0.5 g, 1.26 mmol) in H2O (10 ml) at 70°C. After 8 h, the clear colorless solution was filtered and reduced under vacuum to yield white precipitates. Yield: 449.7 mg (82.8%). Synthesis of bis(3-carboxypropanoato)(trans-(R,R)1,2-diaminocyclohexane)oxalatoplatinum (IV) (2)
Compound 2 was prepared by the addition of succinic anhydride (0.285 g, 2.85 mmol) to the suspension of 1 (0.3 g, 0.696 mmol) in N,N-dimethylformamide (5 ml) and the mixture was stirred at 70°C for 24 h [23]. After
reducing the volume of the solution under vacuum to ∼ 0.5 ml, 10 ml ice acetone was added. The crude product was recrystallized from methanol/acetone to yield a brown solid. Yield: 345.4 mg (78.4%). 1H NMR (500 MHz, DMSO-d6): δ (ppm) 12.08 (bs, 2H), 8.14–8.34 (m, 4H), 2.58 (m, 2H), 2.51 (m, 4H), 2.38 (m, 4H), 2.10 (m, 2H), 1.50 (m, 2H), 1.39 (m, 2H), 1.15 (m, 2H). Electrospray ionization mass spectrometry (ESI-MS; positive mode, m/z): 632.0 [M + H]+, calcd for C16H25N2O12Pt; and 654.0 [M + Na]+, calcd for C16H24N2O12PtNa. Elemental analysis found (calcd) for C16H24N2O12Pt (%): C, 30.35 (30.43); H, 3.78 (3.83); N, 4.72 (4.44). Preparation of conjugates
N-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) were used to react with 2 to form the active ester, which was added gradually to a solution of trastuzumab in PBS (pH = 8.4) and allowed to couple for 16 h at 4°C. The reaction mixture was purified by centrifugal ultrafilters with a cutoff molecular weight of 10 kDa to remove unreacted compound 2. An unrelated IgG was conjugated to 2 as a negative control. The prepared conjugates were analyzed by HPLC (Shimadzu Corp., Kyoto, Japan) using a C18 (4.6 × 250 mm, 5 μm) with UV detection at 280 nm. The mobile phase comprised of 1% ethanoic acid, 1% NaCl, and 98% 0.2 mol/l KH2PO4 at the flow rate of 1.0 ml/min. Platinum concentrations were analyzed by flameless atomic absorption spectrometry. Concentrations of trastuzumab and unrelated IgG were measured using a micro bicinchoninic acid (BCA) protein assay kit. Cell culture
Human cell lines, MDA-MB-231, MCF-7, SK-BR-3, and L-02, were purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). All cell lines were cultured in Dulbecco’s modified Eagle’s medium (high glucose) supplemented with 10% fetal bovine
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Trastuzumab-mediated selective delivery Huang et al. 959
serum and 1% penicillin–streptomycin at 37°C in a humidified incubator with 5% CO2. Cells were detached with trypsin (all from Invitrogen, Carlsbad, California, USA) and plated at the desired cell concentration when grown to 80–90% confluence. Binding affinity to HER2 protein
The binding affinity of trastuzumab–2 conjugates to HER2 protein was determined using an indirect ELISA assay as described previously [24]. Ninety-six-well plates were coated with 0.1 μg/well HER2 protein overnight at 4°C and then blocked with PBS containing 3% bovine serum albumin (BSA) at 37°C for 2 h to avoid nonspecific binding. After washing five times with 0.1% Tween/PBS, 100 μl trastuzumab–2 conjugates, trastuzumab, and unrelated IgG–2 conjugates diluted in 1% BSA with various effective antibody concentrations were added to each well and incubated for 1 h at 37°C. Horseradish peroxidase-conjugated goat anti-human IgG (Fab)′ was added (dilution 1 : 2000) after plate washing and incubated for 1 h at 37°C. Plates were washed as described above, stained with TMB solution at room temperature for 15 min, followed by stopping with 1 mol/l H2SO4. The absorbance was measured at 450 nm.
CCK-8. Oxaliplatin was chosen as a positive reference. Briefly, cells at a density of 5 × 103 per well were plated on 96-well plates for 24 h and then exposed to various concentrations of compounds for 72 h at 37°C. Subsequently, each well was added with 10 μl CCK-8 reagent and the plates were incubated for 2 h, followed by measuring the absorbance at 450 nm. All experiments were conducted in triplicate and the half-maximal inhibitory concentrations (IC50) were calculated. Cell cycle analysis
SK-BR-3 cells were treated with trastuzumab–2 conjugates or oxaliplatin for 48 h and the culture medium was collected carefully. Trypsin was added, and then cells were collected and washed with PBS. After centrifugation, the cell suspensions were fixed by dropwise addition of ice-cold 70% ethanol for 6 h. For staining, cells were centrifuged, resuspended in PBS, and treated with 25 μl PI (100 μg/ml) for 30 min in the dark. The cells were analyzed by a FACS Calibur (BD Biosciences, San Diego, California, USA).
Results and discussion Preparation of conjugates
Binding ability to targeted cells
SK-BR-3 and MDA-MB-231 cells were plated at 5 × 103/well for 24 h at 37°C. After washing with ice-cold PBS, cells were fixed with 4% paraformaldehyde for 20 min at room temperature [25]. Cells were blocked for 2 h with 5% nonfat milk and subsequently incubated with trastuzumab–2 conjugates, trastuzumab, and unrelated IgG–2 conjugates at an effective antibody concentration of 50 μg/well overnight at 37°C. The plates were washed three times with 0.1% Tween/PBS, incubated with FITC-labeled goat anti-human Fab for 1 h in the dark, and then stained with DAPI. The plates were washed three times with PBS and imaged with fluorescence microscopy (Olympus, Tokyo, Japan). DNA-binding study
Trastuzumab–2 conjugates, 2, and oxaliplatin at an effective platinum concentration of 50 μmol/l were incubated with 1 μl PUC19 DNA in the absence or presence of ascorbic acid (AsA, 50 μmol/l) overnight at 37°C. The reaction products were then electrophoresed on 1% agarose gels containing TAE buffer (40 mmol/l Trisacetate, 20 mmol/l sodium acetate, 1 mmol/l EDTA, pH 8.0) for about 1 h at 90 V. DNA bands were captured with Gelred nucleic acid (from Beyotime Institute of Biotechnology, Shanghai, China) and imaged using the Gel Logic 100 Imaging System (Kodak, Rochester, New York, USA). Cytotoxicity
The cytotoxic activity of trastuzumab–2 conjugates and unrelated IgG–2 conjugates was evaluated using a
Two extra ligand sites were introduced to develop octahedral platinum (IV) compound 2, which could be modified for drug-targeting strategies. Trastuzumab–2 conjugates and unrelated IgG–2 conjugates were prepared by coupling 2 to the ε-amino groups of lysine using EDC/NHS chemistry. The designed conjugates facilitate targeting ability for the platinum (IV) drug delivery to HER2 overexpressing cells. Furthermore, corresponding platinum (II) analogs are supposed to be released by intracellular reductants (e.g. AsA) to show anticancer activity. The structure of 2 was characterized by ESI-MS, 1H NMR, and elemental analysis; meanwhile, the conjugates were analyzed by RP-HPLC (Supplementary Fig. S1, Supplemental digital content 1, http://links.lww. com/ACD/A114). A trace of free 2 can be observed at (tR) 9.7 min and the peak for trastuzumab can be observed at 5.5 min, whereas after conjugation, the major peak corresponding to trastuzumab–2 shifts to 4.68 min and almost no peak of free 2 is observed at 9.7 min. These results indicate that 2 has been conjugated to trastuzumab and unreacted 2 has been removed. The antibody concentrations were determined using the BCA assay with BSA as a standard (Supplementary Fig. S2, Supplemental digital content 1, http://links.lww.com/ ACD/A114). In addition, the platinum concentrations were determined by flameless atomic absorption spectrometry and the platinum/antibody ratios were calculated as shown in Table 1. The platinum/antibody ratio is 501 for trastuzumab–2 conjugates and 4.1 for IgG–2 conjugates.
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Anti-Cancer Drugs 2015, Vol 26 No 9
Platinum/antibody ratios of conjugates
Table 1 Conjugate
Antibody concentrationa (mg/ml)
Platinum concentrationb (μg/ml)
Platinum/antibody ratio
1.78 1.56
12.2 8.51
5.1 4.1
Trastuzumab–2 Unrelated IgG–2 a
The antibody concentration was measured using a BCA protein assay kit. The platinum concentration was determined by flameless atomic absorption spectroscopy.
b
Table 2 Half-binding affinity values of trastuzumab and trastuzumab–2 conjugates to HER2 protein
Fig. 2
(a) 1.4 1.2
Trastuzumab Trastuzumab−2 conjugates Unrelated lgG− 2 conjugates
Compound Trastuzumab Trastuzumab–2 conjugates
A450
0.8
Binding ability to targeted cells
0.6 0.4 0.2 0.0 0.1
1
10
100
120 100
100% binding
2.61 ± 0.05 4.91 ± 0.04
HER2, human epidermal growth factor receptor 2.
1.0
(b)
Half-binding affinity values (μg/ml)
80 60 40 20 0 0.1
1 10 Antibody concentration (μg/ml)
100
(a) Superimposed binding curves and (b) binding saturation curves of trastuzumab–2 conjugates or unrelated IgG–2 conjugates to human epidermal growth factor receptor 2 (HER2) protein performed by the enzyme-linked immunosorbent assay. Data are representative of three independent experiments.
Binding affinity to HER2 protein
An indirect ELISA assay, appropriate for evaluation of strongly interacting molecules, was utilized to measure the binding affinities of trastuzumab–2 conjugates and trastuzumab to immobilized HER2 protein [26]. As shown in Fig. 2, trastuzumab–2 conjugates and trastuzumab specifically bind to HER2 protein in a dosedependent manner. The half-binding affinity value for trastuzumab–2 conjugates binding to HER2 is 4.91 μg/ml, which is about two-fold higher than that for unconjugated trastuzumab (Table 2). These results show that, although the affinity is decreased, the trastuzumab–2 conjugates retain the binding ability to HER2. As expected, unrelated IgG–2 conjugates hardly bind to HER2 even at the highest antibody concentration tested.
Trastuzumab–2 conjugates and unrelated IgG–2 conjugates were evaluated for binding ability to SK-BR-3 cells and MDA-MB-231 cells by fluorescence microscopy. Images are shown in Fig. 3. The FITC channel shows the location of trastuzumab or trastuzumab–2 conjugates (binding with FITC-labeled Fab) and the DAPI channel shows the cell nuclei. It is observed from the images that SK-BR-3 cells treated with trastuzumab–2 conjugates or trastuzumab show green fluorescence around the cell surface, whereas no signal is observed in SK-BR-3 cells incubated with unrelated IgG–2 conjugates and in MDA-MB-231 cells incubated with trastuzumab–2 conjugates. These results indicate that the specific binding activity of trastuzumab–2 conjugates to HER2 protein expressed on the surface of SKBR-3 cells is not substantially affected after chemical conjugation, which is consistent with the indication by the ELISA assay.
DNA-binding study
DNA-binding activities of oxaliplatin, 2, and trastuzumab–2 conjugates to PUC19 DNA in the presence and absence of AsA were determined by agarose gel electrophoresis, and the results are shown in Fig. 4. Here, AsA was presented as a reductant. It is known that the binding of platinum complexes to closed-circle DNA can reduce its superhelical density and decrease its rate of migration in the agarose [27]. It is observed that oxaliplatin clearly induces an obvious disappearance of superhelical DNA (form I) and forms a strong band for nicked DNA (form II). 2 formed a weak band for the nicked form, whereas the band formed by trastuzumab–2 conjugates alone is difficult to observe. However, in the presence of AsA, both 2 and trastuzumab–2 conjugates clearly form a strong band for the nicked form, indicating that the reaction products can bind to and unwind PUC19 DNA in a manner similar to oxaliplatin. This result is in agreement with the widely accepted theory that anticancer activity is activated after platinum (IV) drug is reduced to platinum (II) analogs.
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Trastuzumab-mediated selective delivery Huang et al. 961
Fig. 3
FITC
DAPI
Merge
SK-BR-3
Trastuzumab
Trastuzumab− 2 conjugates
MDA-MB-231
Unrelated IgG−2 conjugates
Trastuzumab− 2 conjugates
Individual and merged fluorescence microscopy images of SK-BR-3 and MDA-MB-231 cells after treatment with trastuzumab, trastuzumab–2 conjugates, and IgG–2 conjugates and detected by fluorescein isothiocyanate (FITC)-labeled anti-human antibody and 4′,6-diamidino-2-phenylindole (DAPI).
Cytotoxicity
The cytotoxicity of trastuzumab–2 conjugates was measured in three human breast cancer cell lines, MDAMB-231, MCF-7, and SK-BR-3, which showed low, moderate, and high overexpression of HER2 [19]. Human normal cell line L-02 was selected for comparison. The IC50 values of tested drugs for these cells are listed in Table 3. Oxaliplatin shows promising antiproliferative activity for all tested cell lines, with IC50 values in the low micromolar range (15.4–31.0 μmol/l),
whereas 2 shows lower cytotoxicity, with IC50 values of 21.9 μmol/l for MCF-7 cells, 36.1 μmol/l for MDAMB-231 cells, and 39.6 μmol/l for L-02 cells. Besides, 2 is almost nontoxic against SK-BR-3 cells; the inhibition rate is less than 20% at the highest tested concentration (Supplementary Fig. S3, Supplemental digital content 1, http://links.lww.com/ACD/A114). The decreased cytotoxicity is because of the relative kinetic inertness of the platinum (IV) complex’s octahedral coordination structure. Compared with these two drugs, trastuzumab–2
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962 Anti-Cancer Drugs 2015, Vol 26 No 9
conjugates hardly inhibit the growth of MCF-7, MDAMB-231, and normal cells L-02. The inhibition rate of trastuzumab–2 conjugates against these three cell lines is no more than 20%, even at the highest tested concentration. It is worth noting that trastuzumab–2 conjugates show high cytotoxicity to the HER2-positive SKBR-3 cells, with an IC50 value of 17.2 μmol/l, which is about half of that for oxaliplatin, whereas the unrelated IgG–2 conjugates are much less cytotoxic against SKBR-3 cells than trastuzumab, under the same condition (Supplementary Fig. S3, Supplemental digital content 1, http://links.lww.com/ACD/A114). These results suggest that the cytotoxicity of trastuzumab–2 conjugates is specific and targeting against HER2 overexpressing cells and it is markedly decreased against normal cells compared with free platinum drugs. As a result, it might be able to overcome some side effects related to oxaliplatin.
Fig. 4
1
0
2
3
4
5
Form II
Form I
Agarose gel electrophoresis images of supercoiled PUC19 incubated with platinum complexes. Lane 0, DNA control; lane 1, trastuzumab–2 conjugates; lane 2, trastuzumab–2 conjugates and ascorbic acid (AsA); lane 3, complex 2; lane 4, complex 2 and AsA; lane 5, oxaliplatin.
Table 3 Growth inhibition of different compounds on human breast cancer cells after 72 h IC50 (μmol/l, platinum equivalent)
Cell cycle and apoptosis
Sample
SK-BR-3
MCF-7
MDA-MB-231
L-02
Oxaliplatin 2 Trastuzumab–2 conjugates
31.0 ± 0.1 > 50 17.2 ± 0.5
15.4 ± 0.3 21.9 ± 0.8 > 50
23.1 ± 0.1 36.1 ± 0.7 > 50
28.9 ± 0.7 39.6 ± 1.5 >50
The cell cycle distribution and cell apoptosis after treatment for 48 h were detected using flow cytometry by staining with PI. Oxaliplatin was chosen for comparison. Cell cycle distribution and statistical analysis for different cell cycle phases are shown in Fig. 5. The cells in the
IC50, half-maximal inhibitory concentrations.
Fig. 5
(b) 1600
(c)
Control
Oxaliplatin (50 μmol/l)
Number
1200
Apoptosis Dip G1 Dip G2 Dip S
800
Number
200
400
100
30
90 120 60 Channels (FL2-A)
100
0
0 0
Apoptosis Dip G1 Dip G2 Dip S
150
50
50
0
(d)
Apoptosis Dip G1 Dip G2 Dip S
150
Trastuzumab−2 (5 μmol/l)
200 Number
(a)
0
30
60 90 120 Channels (FL2-A)
0
30
60 90 120 Channels (FL2-A)
160
(e)
400
100
Number
Cell percentage
Trastuzumab−2 (50 μmol/l)
320
Apoptosis Dip G1 Dip G2 Dip S
240 160 80
Control Oxaliplatin (50 μmol/l) Trastuzumab−2 conjugates (5 μmol/l) Trastuzumab−2 conjugates 50 μmol/l)
80 60 40 20 0
0 0
30
60 90 120 Channels (FL2-A)
150
G0/G1
S
G2/M
Apoptosis
Cell cycle distribution analyzed by flow cytometry for different cell cycle phases after treatment with oxaliplatin and trastuzumab–2 conjugates for 48 h.
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Trastuzumab-mediated selective delivery Huang et al. 963
G2/M phase increase after treatment with oxaliplatin; however, cells in the S phase obviously decrease. Trastuzumab–2 conjugates also strongly disturb the cell cycle; cells in the G2/M phase treated with a higher concentration obviously increase, whereas for the S phase, the cell percentage decreases markedly to depletion, probably resulting from the decreased rate of DNA synthesis [28]. This indicates that both oxaliplatin and trastuzumab–2 conjugates significantly arrest the cell cycle mainly at the G2/M phase. Platinum drugs usually induce cancer cells to undergo the apoptosis process, and cells in late apoptosis can be detected by flow cytometry after staining with PI. Trastuzumab–2 conjugates lead to about 32.7% cell apoptosis in 72 h, which is higher than cell apoptosis caused by oxaliplatin (24.8%). Thus, the cell cycle pattern and the cell apoptosis cannot be regulated after conjugation of trastuzumab to the platinum compound 2, suggesting that the platinum group plays the key role in the process of cell killing.
4 5
6 7
8
9
10
11
12
Conclusion In the present study, trastuzumab was conjugated to platinum (IV) complex 2 for the purpose of targeted drug delivery to HER2 overexpressed cells. The platinum/ antibody ratio of synthesized conjugates was calculated to be 5.1 : 1. It was found in this study that trasuzumab–2 conjugates retain their affinity to the HER2 protein, showing specific binding activity to targeted cells. The DNA-binding study of conjugates was investigated, and it was found that platinum (IV)–trastuzumab conjugates were reduced to platinum (II) analogs, followed by binding to and unwinding. Compared with oxaliplatin, the trastuzumab–2 conjugates show markedly improved antiproliferative activity to HER2 overexpressing SK-BR-3 cells, and almost no cytotoxicity to MDA-MB-231, MCF-7, and L-02 cells. Thus, some side effects might be reduced for the conjugates. Similar to oxaliplatin, the trastuzumab–2 conjugates arrest the cell cycle mainly at the G2/M phase, and kill cancer cells in the apoptotic pathway. On the whole, the results in this study can be applied a potential strategy to develop new platinum-based antitumor drugs.
13
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19
20
21
22
Acknowledgements The authors acknowledge financial support from the National Natural Science Foundation of China (nos 21241009 and 21371031) and the International S&T Cooperation Program of China (no. 2015DFG42240).
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Conflicts of interest
There are no conflicts of interest.
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Bose RN. Biomolecular targets for platinum antitumor drugs. Mini Rev Med Chem 2002; 2:103–111. Alessio M, Zanellato I, Bonarrigo I, Gabano E, Ravera M, Osella D. Antiproliferative activity of Pt(IV)-bis(carboxylato) conjugates on malignant pleural mesothelioma cells. J Inorg Biochem 2013; 129:52–57. Shi Y, Liu SA, Kerwood DJ, Goodisman J, Dabrowiak JC. Pt(IV) complexes as prodrugs for cisplatin. J Inorg Biochem 2012; 107:6–14. Zhang JZ, Wexselblatt E, Hambley TW, Gibson D. Pt(IV) analogs of oxaliplatin that do not follow the expected correlation between electrochemical reduction potential and rate of reduction by ascorbate. Chem Commun 2012; 48:847–849. Yang J, Sun X, Mao W, Sui M, Tang J, Shen Y. Conjugate of Pt(IV)-histone deacetylase inhibitor as a prodrug for cancer chemotherapy. Mol Pharm 2012; 9:2793–2800. Dhar S, Gu FX, Langer R, Farokhzad OC, Lippard SJ. Targeted delivery of cisplatin to prostate cancer cells by aptamer functionalized Pt(IV) prodrugPLGA-PEG nanoparticles. Proc Natl Acad Sci USA 2008; 105:17356–17361. Graf N, Mokhtari TE, Papayannopoulos IA, Lippard SJ. Platinum(IV)chlorotoxin (CTX) conjugates for targeting cancer cells. J Inorg Biochem 2012; 110:58–63. Dhar S, Daniel WL, Giljohann DA, Mirkin CA, Lippard SJ. Polyvalent oligonucleotide gold nanoparticle conjugates as delivery vehicles for platinum(IV) warheads. J Am Chem Soc 2009; 131:14652–14653. Barnes KR, Kutikov A, Lippard SJ. Synthesis, characterization, and cytotoxicity of a series of estrogen-tethered platinum(IV) complexes. Chem Biol 2004; 11:557–564. Gabano E, Ravera M, Cassino C, Bonetti S, Palmisano G, Osella D. Stepwise assembly of platinum-folic acid conjugates. Inorganica Chim Acta 2008; 361:1447–1455. Ndinguri MW, Solipuram R, Gambrell RP, Aggarwal S, Hammer RP. Peptide targeting of platinum anti-cancer drugs. Bioconjug Chem 2009; 20:1869–1878. Mukhopadhyay S, Barnés CM, Haskel A, Short SM, Barnes KR, Lippard SJ. Conjugated platinum (IV)-peptide complexes for targeting angiogenic tumor vasculature. Bioconjug Chem 2008; 19:39–49. Flygare JA, Pillow TH, Aristoff P. Antibody–drug conjugates for the treatment of cancer. Chem Biol Drug Des 2013; 81:113–121. Stack GD, Walsh JJ. Optimising the delivery of tubulin targeting agents through antibody conjugation. Pharm Res 2012; 29:2972–2984. Steichen SD, Caldorera-Moore M, Peppas NA. A review of current nanoparticle and targeting moieties for the delivery of cancer therapeutics. Eur J Pharm Sci 2013; 48:416–427. Gianolio DA, Rouleau C, Bauta WE, Lovett D, Cantrell WR Jr, Recio A 3rd, et al. Targeting HER2-positive cancer with dolastatin 15 derivatives conjugated to trastuzumab, novel antibody-drug conjugates. Cancer Chemother Pharmacol 2012; 70:439–449. Mi Y, Zhao J, Feng SS. Targeted co-delivery of docetaxel, cisplatin and herceptin by vitamin E TPGS–cisplatin prodrug nanoparticles for multimodality treatment of cancer. J Control Release 2013; 169:185–192. Xiao H, Song H, Yang Q, Cai H, Qi R, Yan L, et al. A prodrug strategy to deliver cisplatin(IV) and paclitaxel in nanomicelles to improve efficacy and tolerance. Biomaterials 2012; 33:6507–6519. Khan SRA, Huang S, Shamsuddin S, Inutsuka S, Whitmire KH, Siddik ZH, et al. Synthesis, characterization and cytotoxicity of new platinum (IV) axial carboxylate complexes: crystal structure of potential antitumor agent [PtIV (trans-1R,2R-diaminocyclohexane)trans(acetate)2Cl2]. Bioorg Med Chem 2000; 8:515–521. Reithofer MR, Valiahdi SM, Jakupec MA, Arion VB, Egger A, Galanski M, Keppler BK. Novel di- and tetracarboxylatoplatinum(IV) complexes. Synthesis, characterization, cytotoxic activity, and DNA platination. J Med Chem 2007; 50:6692–6699. Chen C, Constantinou A, Chester KA, Vyas B, Canis K, Haslam SM, et al. Glycoengineering approach to half-life extension of recombinant biotherapeutics. Bioconjug Chem 2012; 23:1524–1533. Chen R, Zhang D, Mao Y, Zhu J, Ming H, Wen J, et al. A human Fab-based immunoconjugate specific for the LMP1 extracellular domain inhibits nasopharyngeal carcinoma growth in vitro and in vivo. Mol Cancer Ther 2012; 11:594–603. Khalili H, Godwin A, Choi JW, Lever R, Brocchini S. Comparative binding of disulfide-bridged PEG-Fabs. Bioconjug Chem 2012; 23:2262–2277. Wu S, Wang X, He Y, Zhu Z, Zhu C, Guo Z. A monofunctional trinuclear platinum complex with steric hindrance demonstrates strong cytotoxicity against tumor cells. J Inorg Biochem 2014; 139:77–84. Volckova E, Weaver E, Bose RN. Insight into the reactive form of the anticancer agent iproplatin. Eur J Med Chem 2008; 43:1081–1084.
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Trastuzumab-mediated selective delivery for platinum drug to HER2-positive breast cancer cells Rong Huanga, Yu Suna,b, Qihe Gaoc, Qiucui Wanga and Baiwang Suna Oxaliplatin is used widely as an anticancer drug for clinical treatment. However, its applications are limited because of its poor selectivity. In this work, we described the design, synthesis, and characterization of conjugates combining trastuzumab with a platinum (IV) analog of oxaliplatin, in which the trastuzumab acted as an active targeting agent for HER2-positive cancer cells. Indirect enzyme-linked immunosorbent assay and immunofluorescence study indicated the platinum (IV)–trastuzumab conjugates retained specific binding activity to HER2 overexpressed SK-BR-3 cells. In the presence of ascorbic acid, platinum (IV)–trastuzumab conjugates were reduced to platinum (II) analogs, which could bind to and unwind PUC19 DNA in a manner similar to oxaliplatin. The cytotoxic study was tested on three breast cell lines: SK-BR-3, MCF-7, and MDAMB-231. Platinum (IV)–trastuzumab conjugates showed promising antiproliferative activity against SK-BR-3 cells,
Introduction Platinum-based antitumor drugs are used widely for the treatment of various malignancies such as genitourinary, colorectal, and non-small-cell lung cancers [1,2]. Compared with cisplatin and carboplatin, oxaliplatin (Fig. 1) has a slightly different mode of action. The chelating oxalate ligand is more inert as a leaving group compared with chlorides, lowering the aquation rate and avoiding drug resistance [3]. Oxaliplatin is highly active in colorectal cancer and has shown very promising activity in metastatic breast cancer either in monotherapy or in combination with 5-fluorouracil or trastuzumab [4]. However, the application of oxaliplatin is limited by severe side effects, such as nephrotoxicity, ototoxicity, and neurotoxicity, and its poor ability to distinguish healthy and cancer cells. As a result, there has been considerable focus on the development of targeting delivery for platinum drugs. It is widely accepted that platinum (IV) compounds act as potential prodrugs, which are believed to be activated in the cancer cells by being reduced to platinum (II) analogs [5–7]. The platinum (IV) compounds, with an octahedral coordination structure, are kinetically inert compared with platinum (II) compounds [8]. The two extra ligands introduced into the platinum center can be modified for drug-targeting strategies [9–11], which are lost during Supplemental digital content is available for this article. Direct URL citations appear in the printed text and are provided in the HTML and PDF versions of this article on the journal's website (www.anti-cancerdrugs.com). 0959-4973 Copyright © 2015 Wolters Kluwer Health, Inc. All rights reserved.
but significantly decreased the inhibition to MDA-MB-231 and MCF-7 cells. The flow cytometric analysis showed that the conjugates arrested the cell cycle mainly at the G2/M phase and killed the cells through an apoptotic pathway. Anti-Cancer Drugs 26:957–963 Copyright © 2015 Wolters Kluwer Health, Inc. All rights reserved. Anti-Cancer Drugs 2015, 26:957–963 Keywords: antibody drug conjugate, anticancer drug, cell cycle arrest, platinum (IV) drug, targeting delivery a
School of Chemistry and Chemical Engineering, Southeast University, Nanjing, School of Pharmacy, Wannan Medical College, Wuhu and cInstitute of Microbiology, Chinese Academy of Science, Beijing, People’s Republic of China b
Correspondence to Baiwang Sun, School of Chemistry and Chemical Engineering, Southeast University, Nanjing 211189, People’s Republic of China Tel/fax: + 86 25 5209 0614; e-mail: [email protected] Received 26 March 2015 Revised form accepted 23 June 2015
reduction and are not involved in the mechanism of cell killing. Folic acid, estrogens, aptamers, and peptides have already been reported as targeting agents for platinum drugs delivery [12–15]. For the past few years, therapeutic antibodies have been considered promising targeting agents because of their low immunogenicity and high affinity to cancer cells [16,17]. Trastuzumab is a humanized monoclonal antibody that can specifically bind to the cell surface receptor, human epidermal growth factor receptor 2 (HER2). It has been considered a successful targeting agent for anticancer drugs delivery [18–20]. Thus, we chose trastuzumab as a representative antibody in our study. In this work, we designed a drug delivery strategy based on the synthesized platinum (IV) compound 2 (Fig. 1) using trastuzumab as an active cancer targeting agent. The conjugates are supposed to retain the specific binding ability to targeted cancer cells and confer additional cell-killing activity after reducing to platinum (II) analogs in cells. To test this assumption, three breast cell lines with different HER2 expression levels, SK-BR-3, MCF-7, and MDAMB-231, were selected. Indirect enzyme-linked immunosorbent assay (ELISA) assay, immunofluorescence study, and cytotoxicity study were carried out in this study to investigate the targeting binding activity and cytotoxicity of conjugates. Furthermore, gel electrophoresis and flow cytometry were used to discuss the DNA binding and cellkilling mode of conjugates. DOI: 10.1097/CAD.0000000000000272
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Fig. 1
O OH O
OH H2 N
O
O
H2 N
O
Pt N H2
H2 N
O
O
N H2
O
O
O
O
Pt
Pt O
O
O
O
O
N H2 O
OH HO Oxaliplatin
O
1
2
Structures of oxaliplatin, 1 and 2.
Materials and methods Materials
Oxaliplatin was obtained from Shangdong Boyuan Pharmaceutical Co. Ltd (Shangdong, China). Trastuzumab (10 mg/ml) was purchased from Harbin Pharmaceutical Group Bioengineering Co. Ltd (Harbin, China). All other chemicals were of analytical grade and used without further purification. HER2 protein was purchased from R&D Systems Inc. (Minneapolis, Minnesota, USA). Horseradish peroxidase-conjugated goat anti-human IgG (Fab)′, fluorescein isothiocyanate (FITC)-labeled goat anti-human IgG (Fab)′, and 4′,6-diamidino-2-phenylindole (DAPI) were purchased from Sigma-Aldrich (St. Louis, Missouri, USA). Cell counting kit-8 (CCK-8) was obtained from Dojindo Molecular Technologies (Kumamoto, Japan). 3,3′,5,5′Tetramethylbenzidine (TMB) solution was obtained from the Beyotime Institute of Biotechnology (Shanghai, China). The PUC19 plasmid DNA substrate was purchased from New England Biolabs (Ipswich, Massachusetts, USA) and propidium iodide (PI) was purchased from Becton Dickinson and Company (Franklin Lakes, New Jersey, USA). Preparation of trastuzumab–2 conjugates Synthesis of trans-R,R-1,2-diaminocyclohexane dihydroxo(oxalate)platinum (IV) (1)
Compound 1 was synthesized in a similar manner according to the literature procedures [21,22]. Briefly, hydrogen peroxide (30 wt%, 20 ml) was added dropwise to a suspension of oxaliplatin (0.5 g, 1.26 mmol) in H2O (10 ml) at 70°C. After 8 h, the clear colorless solution was filtered and reduced under vacuum to yield white precipitates. Yield: 449.7 mg (82.8%). Synthesis of bis(3-carboxypropanoato)(trans-(R,R)1,2-diaminocyclohexane)oxalatoplatinum (IV) (2)
Compound 2 was prepared by the addition of succinic anhydride (0.285 g, 2.85 mmol) to the suspension of 1 (0.3 g, 0.696 mmol) in N,N-dimethylformamide (5 ml) and the mixture was stirred at 70°C for 24 h [23]. After
reducing the volume of the solution under vacuum to ∼ 0.5 ml, 10 ml ice acetone was added. The crude product was recrystallized from methanol/acetone to yield a brown solid. Yield: 345.4 mg (78.4%). 1H NMR (500 MHz, DMSO-d6): δ (ppm) 12.08 (bs, 2H), 8.14–8.34 (m, 4H), 2.58 (m, 2H), 2.51 (m, 4H), 2.38 (m, 4H), 2.10 (m, 2H), 1.50 (m, 2H), 1.39 (m, 2H), 1.15 (m, 2H). Electrospray ionization mass spectrometry (ESI-MS; positive mode, m/z): 632.0 [M + H]+, calcd for C16H25N2O12Pt; and 654.0 [M + Na]+, calcd for C16H24N2O12PtNa. Elemental analysis found (calcd) for C16H24N2O12Pt (%): C, 30.35 (30.43); H, 3.78 (3.83); N, 4.72 (4.44). Preparation of conjugates
N-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) were used to react with 2 to form the active ester, which was added gradually to a solution of trastuzumab in PBS (pH = 8.4) and allowed to couple for 16 h at 4°C. The reaction mixture was purified by centrifugal ultrafilters with a cutoff molecular weight of 10 kDa to remove unreacted compound 2. An unrelated IgG was conjugated to 2 as a negative control. The prepared conjugates were analyzed by HPLC (Shimadzu Corp., Kyoto, Japan) using a C18 (4.6 × 250 mm, 5 μm) with UV detection at 280 nm. The mobile phase comprised of 1% ethanoic acid, 1% NaCl, and 98% 0.2 mol/l KH2PO4 at the flow rate of 1.0 ml/min. Platinum concentrations were analyzed by flameless atomic absorption spectrometry. Concentrations of trastuzumab and unrelated IgG were measured using a micro bicinchoninic acid (BCA) protein assay kit. Cell culture
Human cell lines, MDA-MB-231, MCF-7, SK-BR-3, and L-02, were purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). All cell lines were cultured in Dulbecco’s modified Eagle’s medium (high glucose) supplemented with 10% fetal bovine
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Trastuzumab-mediated selective delivery Huang et al. 959
serum and 1% penicillin–streptomycin at 37°C in a humidified incubator with 5% CO2. Cells were detached with trypsin (all from Invitrogen, Carlsbad, California, USA) and plated at the desired cell concentration when grown to 80–90% confluence. Binding affinity to HER2 protein
The binding affinity of trastuzumab–2 conjugates to HER2 protein was determined using an indirect ELISA assay as described previously [24]. Ninety-six-well plates were coated with 0.1 μg/well HER2 protein overnight at 4°C and then blocked with PBS containing 3% bovine serum albumin (BSA) at 37°C for 2 h to avoid nonspecific binding. After washing five times with 0.1% Tween/PBS, 100 μl trastuzumab–2 conjugates, trastuzumab, and unrelated IgG–2 conjugates diluted in 1% BSA with various effective antibody concentrations were added to each well and incubated for 1 h at 37°C. Horseradish peroxidase-conjugated goat anti-human IgG (Fab)′ was added (dilution 1 : 2000) after plate washing and incubated for 1 h at 37°C. Plates were washed as described above, stained with TMB solution at room temperature for 15 min, followed by stopping with 1 mol/l H2SO4. The absorbance was measured at 450 nm.
CCK-8. Oxaliplatin was chosen as a positive reference. Briefly, cells at a density of 5 × 103 per well were plated on 96-well plates for 24 h and then exposed to various concentrations of compounds for 72 h at 37°C. Subsequently, each well was added with 10 μl CCK-8 reagent and the plates were incubated for 2 h, followed by measuring the absorbance at 450 nm. All experiments were conducted in triplicate and the half-maximal inhibitory concentrations (IC50) were calculated. Cell cycle analysis
SK-BR-3 cells were treated with trastuzumab–2 conjugates or oxaliplatin for 48 h and the culture medium was collected carefully. Trypsin was added, and then cells were collected and washed with PBS. After centrifugation, the cell suspensions were fixed by dropwise addition of ice-cold 70% ethanol for 6 h. For staining, cells were centrifuged, resuspended in PBS, and treated with 25 μl PI (100 μg/ml) for 30 min in the dark. The cells were analyzed by a FACS Calibur (BD Biosciences, San Diego, California, USA).
Results and discussion Preparation of conjugates
Binding ability to targeted cells
SK-BR-3 and MDA-MB-231 cells were plated at 5 × 103/well for 24 h at 37°C. After washing with ice-cold PBS, cells were fixed with 4% paraformaldehyde for 20 min at room temperature [25]. Cells were blocked for 2 h with 5% nonfat milk and subsequently incubated with trastuzumab–2 conjugates, trastuzumab, and unrelated IgG–2 conjugates at an effective antibody concentration of 50 μg/well overnight at 37°C. The plates were washed three times with 0.1% Tween/PBS, incubated with FITC-labeled goat anti-human Fab for 1 h in the dark, and then stained with DAPI. The plates were washed three times with PBS and imaged with fluorescence microscopy (Olympus, Tokyo, Japan). DNA-binding study
Trastuzumab–2 conjugates, 2, and oxaliplatin at an effective platinum concentration of 50 μmol/l were incubated with 1 μl PUC19 DNA in the absence or presence of ascorbic acid (AsA, 50 μmol/l) overnight at 37°C. The reaction products were then electrophoresed on 1% agarose gels containing TAE buffer (40 mmol/l Trisacetate, 20 mmol/l sodium acetate, 1 mmol/l EDTA, pH 8.0) for about 1 h at 90 V. DNA bands were captured with Gelred nucleic acid (from Beyotime Institute of Biotechnology, Shanghai, China) and imaged using the Gel Logic 100 Imaging System (Kodak, Rochester, New York, USA). Cytotoxicity
The cytotoxic activity of trastuzumab–2 conjugates and unrelated IgG–2 conjugates was evaluated using a
Two extra ligand sites were introduced to develop octahedral platinum (IV) compound 2, which could be modified for drug-targeting strategies. Trastuzumab–2 conjugates and unrelated IgG–2 conjugates were prepared by coupling 2 to the ε-amino groups of lysine using EDC/NHS chemistry. The designed conjugates facilitate targeting ability for the platinum (IV) drug delivery to HER2 overexpressing cells. Furthermore, corresponding platinum (II) analogs are supposed to be released by intracellular reductants (e.g. AsA) to show anticancer activity. The structure of 2 was characterized by ESI-MS, 1H NMR, and elemental analysis; meanwhile, the conjugates were analyzed by RP-HPLC (Supplementary Fig. S1, Supplemental digital content 1, http://links.lww. com/ACD/A114). A trace of free 2 can be observed at (tR) 9.7 min and the peak for trastuzumab can be observed at 5.5 min, whereas after conjugation, the major peak corresponding to trastuzumab–2 shifts to 4.68 min and almost no peak of free 2 is observed at 9.7 min. These results indicate that 2 has been conjugated to trastuzumab and unreacted 2 has been removed. The antibody concentrations were determined using the BCA assay with BSA as a standard (Supplementary Fig. S2, Supplemental digital content 1, http://links.lww.com/ ACD/A114). In addition, the platinum concentrations were determined by flameless atomic absorption spectrometry and the platinum/antibody ratios were calculated as shown in Table 1. The platinum/antibody ratio is 501 for trastuzumab–2 conjugates and 4.1 for IgG–2 conjugates.
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Anti-Cancer Drugs 2015, Vol 26 No 9
Platinum/antibody ratios of conjugates
Table 1 Conjugate
Antibody concentrationa (mg/ml)
Platinum concentrationb (μg/ml)
Platinum/antibody ratio
1.78 1.56
12.2 8.51
5.1 4.1
Trastuzumab–2 Unrelated IgG–2 a
The antibody concentration was measured using a BCA protein assay kit. The platinum concentration was determined by flameless atomic absorption spectroscopy.
b
Table 2 Half-binding affinity values of trastuzumab and trastuzumab–2 conjugates to HER2 protein
Fig. 2
(a) 1.4 1.2
Trastuzumab Trastuzumab−2 conjugates Unrelated lgG− 2 conjugates
Compound Trastuzumab Trastuzumab–2 conjugates
A450
0.8
Binding ability to targeted cells
0.6 0.4 0.2 0.0 0.1
1
10
100
120 100
100% binding
2.61 ± 0.05 4.91 ± 0.04
HER2, human epidermal growth factor receptor 2.
1.0
(b)
Half-binding affinity values (μg/ml)
80 60 40 20 0 0.1
1 10 Antibody concentration (μg/ml)
100
(a) Superimposed binding curves and (b) binding saturation curves of trastuzumab–2 conjugates or unrelated IgG–2 conjugates to human epidermal growth factor receptor 2 (HER2) protein performed by the enzyme-linked immunosorbent assay. Data are representative of three independent experiments.
Binding affinity to HER2 protein
An indirect ELISA assay, appropriate for evaluation of strongly interacting molecules, was utilized to measure the binding affinities of trastuzumab–2 conjugates and trastuzumab to immobilized HER2 protein [26]. As shown in Fig. 2, trastuzumab–2 conjugates and trastuzumab specifically bind to HER2 protein in a dosedependent manner. The half-binding affinity value for trastuzumab–2 conjugates binding to HER2 is 4.91 μg/ml, which is about two-fold higher than that for unconjugated trastuzumab (Table 2). These results show that, although the affinity is decreased, the trastuzumab–2 conjugates retain the binding ability to HER2. As expected, unrelated IgG–2 conjugates hardly bind to HER2 even at the highest antibody concentration tested.
Trastuzumab–2 conjugates and unrelated IgG–2 conjugates were evaluated for binding ability to SK-BR-3 cells and MDA-MB-231 cells by fluorescence microscopy. Images are shown in Fig. 3. The FITC channel shows the location of trastuzumab or trastuzumab–2 conjugates (binding with FITC-labeled Fab) and the DAPI channel shows the cell nuclei. It is observed from the images that SK-BR-3 cells treated with trastuzumab–2 conjugates or trastuzumab show green fluorescence around the cell surface, whereas no signal is observed in SK-BR-3 cells incubated with unrelated IgG–2 conjugates and in MDA-MB-231 cells incubated with trastuzumab–2 conjugates. These results indicate that the specific binding activity of trastuzumab–2 conjugates to HER2 protein expressed on the surface of SKBR-3 cells is not substantially affected after chemical conjugation, which is consistent with the indication by the ELISA assay.
DNA-binding study
DNA-binding activities of oxaliplatin, 2, and trastuzumab–2 conjugates to PUC19 DNA in the presence and absence of AsA were determined by agarose gel electrophoresis, and the results are shown in Fig. 4. Here, AsA was presented as a reductant. It is known that the binding of platinum complexes to closed-circle DNA can reduce its superhelical density and decrease its rate of migration in the agarose [27]. It is observed that oxaliplatin clearly induces an obvious disappearance of superhelical DNA (form I) and forms a strong band for nicked DNA (form II). 2 formed a weak band for the nicked form, whereas the band formed by trastuzumab–2 conjugates alone is difficult to observe. However, in the presence of AsA, both 2 and trastuzumab–2 conjugates clearly form a strong band for the nicked form, indicating that the reaction products can bind to and unwind PUC19 DNA in a manner similar to oxaliplatin. This result is in agreement with the widely accepted theory that anticancer activity is activated after platinum (IV) drug is reduced to platinum (II) analogs.
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Trastuzumab-mediated selective delivery Huang et al. 961
Fig. 3
FITC
DAPI
Merge
SK-BR-3
Trastuzumab
Trastuzumab− 2 conjugates
MDA-MB-231
Unrelated IgG−2 conjugates
Trastuzumab− 2 conjugates
Individual and merged fluorescence microscopy images of SK-BR-3 and MDA-MB-231 cells after treatment with trastuzumab, trastuzumab–2 conjugates, and IgG–2 conjugates and detected by fluorescein isothiocyanate (FITC)-labeled anti-human antibody and 4′,6-diamidino-2-phenylindole (DAPI).
Cytotoxicity
The cytotoxicity of trastuzumab–2 conjugates was measured in three human breast cancer cell lines, MDAMB-231, MCF-7, and SK-BR-3, which showed low, moderate, and high overexpression of HER2 [19]. Human normal cell line L-02 was selected for comparison. The IC50 values of tested drugs for these cells are listed in Table 3. Oxaliplatin shows promising antiproliferative activity for all tested cell lines, with IC50 values in the low micromolar range (15.4–31.0 μmol/l),
whereas 2 shows lower cytotoxicity, with IC50 values of 21.9 μmol/l for MCF-7 cells, 36.1 μmol/l for MDAMB-231 cells, and 39.6 μmol/l for L-02 cells. Besides, 2 is almost nontoxic against SK-BR-3 cells; the inhibition rate is less than 20% at the highest tested concentration (Supplementary Fig. S3, Supplemental digital content 1, http://links.lww.com/ACD/A114). The decreased cytotoxicity is because of the relative kinetic inertness of the platinum (IV) complex’s octahedral coordination structure. Compared with these two drugs, trastuzumab–2
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962 Anti-Cancer Drugs 2015, Vol 26 No 9
conjugates hardly inhibit the growth of MCF-7, MDAMB-231, and normal cells L-02. The inhibition rate of trastuzumab–2 conjugates against these three cell lines is no more than 20%, even at the highest tested concentration. It is worth noting that trastuzumab–2 conjugates show high cytotoxicity to the HER2-positive SKBR-3 cells, with an IC50 value of 17.2 μmol/l, which is about half of that for oxaliplatin, whereas the unrelated IgG–2 conjugates are much less cytotoxic against SKBR-3 cells than trastuzumab, under the same condition (Supplementary Fig. S3, Supplemental digital content 1, http://links.lww.com/ACD/A114). These results suggest that the cytotoxicity of trastuzumab–2 conjugates is specific and targeting against HER2 overexpressing cells and it is markedly decreased against normal cells compared with free platinum drugs. As a result, it might be able to overcome some side effects related to oxaliplatin.
Fig. 4
1
0
2
3
4
5
Form II
Form I
Agarose gel electrophoresis images of supercoiled PUC19 incubated with platinum complexes. Lane 0, DNA control; lane 1, trastuzumab–2 conjugates; lane 2, trastuzumab–2 conjugates and ascorbic acid (AsA); lane 3, complex 2; lane 4, complex 2 and AsA; lane 5, oxaliplatin.
Table 3 Growth inhibition of different compounds on human breast cancer cells after 72 h IC50 (μmol/l, platinum equivalent)
Cell cycle and apoptosis
Sample
SK-BR-3
MCF-7
MDA-MB-231
L-02
Oxaliplatin 2 Trastuzumab–2 conjugates
31.0 ± 0.1 > 50 17.2 ± 0.5
15.4 ± 0.3 21.9 ± 0.8 > 50
23.1 ± 0.1 36.1 ± 0.7 > 50
28.9 ± 0.7 39.6 ± 1.5 >50
The cell cycle distribution and cell apoptosis after treatment for 48 h were detected using flow cytometry by staining with PI. Oxaliplatin was chosen for comparison. Cell cycle distribution and statistical analysis for different cell cycle phases are shown in Fig. 5. The cells in the
IC50, half-maximal inhibitory concentrations.
Fig. 5
(b) 1600
(c)
Control
Oxaliplatin (50 μmol/l)
Number
1200
Apoptosis Dip G1 Dip G2 Dip S
800
Number
200
400
100
30
90 120 60 Channels (FL2-A)
100
0
0 0
Apoptosis Dip G1 Dip G2 Dip S
150
50
50
0
(d)
Apoptosis Dip G1 Dip G2 Dip S
150
Trastuzumab−2 (5 μmol/l)
200 Number
(a)
0
30
60 90 120 Channels (FL2-A)
0
30
60 90 120 Channels (FL2-A)
160
(e)
400
100
Number
Cell percentage
Trastuzumab−2 (50 μmol/l)
320
Apoptosis Dip G1 Dip G2 Dip S
240 160 80
Control Oxaliplatin (50 μmol/l) Trastuzumab−2 conjugates (5 μmol/l) Trastuzumab−2 conjugates 50 μmol/l)
80 60 40 20 0
0 0
30
60 90 120 Channels (FL2-A)
150
G0/G1
S
G2/M
Apoptosis
Cell cycle distribution analyzed by flow cytometry for different cell cycle phases after treatment with oxaliplatin and trastuzumab–2 conjugates for 48 h.
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Trastuzumab-mediated selective delivery Huang et al. 963
G2/M phase increase after treatment with oxaliplatin; however, cells in the S phase obviously decrease. Trastuzumab–2 conjugates also strongly disturb the cell cycle; cells in the G2/M phase treated with a higher concentration obviously increase, whereas for the S phase, the cell percentage decreases markedly to depletion, probably resulting from the decreased rate of DNA synthesis [28]. This indicates that both oxaliplatin and trastuzumab–2 conjugates significantly arrest the cell cycle mainly at the G2/M phase. Platinum drugs usually induce cancer cells to undergo the apoptosis process, and cells in late apoptosis can be detected by flow cytometry after staining with PI. Trastuzumab–2 conjugates lead to about 32.7% cell apoptosis in 72 h, which is higher than cell apoptosis caused by oxaliplatin (24.8%). Thus, the cell cycle pattern and the cell apoptosis cannot be regulated after conjugation of trastuzumab to the platinum compound 2, suggesting that the platinum group plays the key role in the process of cell killing.
4 5
6 7
8
9
10
11
12
Conclusion In the present study, trastuzumab was conjugated to platinum (IV) complex 2 for the purpose of targeted drug delivery to HER2 overexpressed cells. The platinum/ antibody ratio of synthesized conjugates was calculated to be 5.1 : 1. It was found in this study that trasuzumab–2 conjugates retain their affinity to the HER2 protein, showing specific binding activity to targeted cells. The DNA-binding study of conjugates was investigated, and it was found that platinum (IV)–trastuzumab conjugates were reduced to platinum (II) analogs, followed by binding to and unwinding. Compared with oxaliplatin, the trastuzumab–2 conjugates show markedly improved antiproliferative activity to HER2 overexpressing SK-BR-3 cells, and almost no cytotoxicity to MDA-MB-231, MCF-7, and L-02 cells. Thus, some side effects might be reduced for the conjugates. Similar to oxaliplatin, the trastuzumab–2 conjugates arrest the cell cycle mainly at the G2/M phase, and kill cancer cells in the apoptotic pathway. On the whole, the results in this study can be applied a potential strategy to develop new platinum-based antitumor drugs.
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Acknowledgements The authors acknowledge financial support from the National Natural Science Foundation of China (nos 21241009 and 21371031) and the International S&T Cooperation Program of China (no. 2015DFG42240).
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Conflicts of interest
There are no conflicts of interest.
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