698
Preclinical report
A hybrid platinum drug dichloroacetate-platinum(II) overcomes cisplatin drug resistance through dual organelle targeting Yu Zhanga,*, Guannan Guob,*, Ben Maa, Rong Dua, Haihua Xiaoc, Xiaoguang Yangb, Wenliang Lia, Ying Gaod, Yuxin Lib and Xiabin Jingc A hybrid drug dichloroacetate-platinum(II) [DCA-Pt(II)] was found to overcome cisplatin drug resistance of ovarian cancer through a dual targeting mode, which is different from the mode of action of the present platinum (Pt) drugs used in clinics. DCA-Pt(II) exhibited remarkable cytotoxicity against both cisplatin-sensitive (A2780) and cisplatinresistant (A2780DDP) ovarian cancer cells. The Pt and Pt-DNA adduct content test showed that there was less Pt cellular uptake and fewer Pt-DNA adducts were present after DCA-Pt(II) treatment compared with treatment with cisplatin, carboplatin, and some other drugs. In the study, the effects of DCA-Pt(II) on the cell cycle and apoptosis were also investigated, which showed that DCA-Pt(II) induced G2/M phase arrest and mitochondria-mediated apoptosis in both sensitive and resistant cells lines. Interestingly, DCA-Pt(II) had much greater effects on mitochondria in A2780DDP cell lines than in A2780 cell lines. Anti-Cancer Drugs 26:698–705 Copyright © 2015 Wolters Kluwer Health, Inc. All rights reserved.
Introduction Ovarian cancer is the most common cause of gynecologic neoplasms and the fifth most common cause of cancer mortality among women worldwide [1]. The mortality rate among women with ovarian cancer, especially for epithelial ovarian cancer (85% of all cases of ovarian cancer), is quite high [1,2]. Despite marked progress in surgical techniques, as well as in treatment options, the 5-year survival rate for stage III/IV ovarian cancer still remains at ∼ 45% [1,2]. Cisplatin is a DNA alkylating agent [3,4] and has been the most active drug for ovarian cancer for decades [4,5]. However, although most women with ovarian cancer respond to frontline platinum (Pt) drugs, the majority of them develop tumors resistant to Pt drugs [5,6]. Resistance to Pt drugs falls into two major groups: (i) limited Pt-DNA adduct formation (reduced drug internalization and enhanced detoxification through thiols, proteins, etc.) and (ii) cell signaling pathway alteration, which prevents cell death from formation of Pt-DNA adducts (increased Pt-DNA damage tolerance or enhanced repair of damaged DNA) [4–6].
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.
Anti-Cancer Drugs 2015, 26:698–705 Keywords: antitumor, dichloroacetate, drug resistance, organelle targeting, platinum drug a
National Engineering Laboratory for Druggable Gene and Protein Screening, Jilin Province Key Laboratory on Chemistry and Biology of Natural Drugs in Changbai Mountain, School of Life Sciences, Northeast Normal University, cState Key Laboratory of Polymer Physics and Chemistry, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences and dSchool of Chemistry & Environmental Engineering, Changchun University of Science and Technology, Changchun, China b
Correspondence to Wenliang Li, PhD, National Engineering Laboratory for Druggable Gene and Protein Screening, Northeast Normal University, Changchun 130024, China Tel: + 86 431 89165937; fax: + 86 431 89165917; e-mail: [email protected] and Yuxin Li, PhD, Jilin Province Key Laboratory on Chemistry and Biology of Natural Drugs in Changbai Mountain, School of Life Sciences, Northeast Normal University, Changchun 130024, China Tel/fax:+ 86 431 89165926; e-mail: [email protected] *Yu Zhang and Guannan Guo contributed equally to the writing of this article. Received 31 October 2014 Revised form accepted 24 February 2015
Cell targeting is the key to cancer treatment [7,8]. The mitochondrion is the power plant of cells, providing energy for various cellular activities. Tumor cells have altered metabolism through conversion of glucose oxidation to glycolysis. The excessive glycolysis results in hypoxia, evading apoptosis and leading to unlimited proliferation. Moreover, cancer cells have a hyperpolarized mitochondrial membrane potential (MMP, Δψm). Loss of Δψm is an early event in apoptotic cascades [9–11]. Therefore, the mitochondria might be a potential cancer target. Dichloroacetate (DCA) can enhance apoptosis by increasing the movement of pyruvate into the mitochondria, shifting the metabolism from glycolysis to glucose oxidation and decreasing Δψm. This eventually leads to an increase in apoptosis and a decrease in cellular proliferation [12,13]. Thus, DCA causes cancer regression by sensitizing cells to chemotherapy through the mitochondrial apoptotic pathway, with minimal effects on normal cells [14]. A series of Pt drugs were developed by our group [14,15]. Among them, a hybrid Pt(II) drug, DCA-Pt(II), with both DCA and Pt in one molecule [15] showed a concerted mode of action and good efficacy in tumor inhibition. We envisage here that DCA-Pt(II) can overcome cisplatin drug resistance through dual targeting organelles. This drug can undergo subsequent dissociation and hydration after DOI: 10.1097/CAD.0000000000000234
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DCA-Pt(II) overcomes cisplatin drug resistance Zhang et al. 699
internalization by the cancer cells because of the lower chloride concentrations (from ∼ 100 to 2–10 mmol/l) and lower pH values (pH = 7.4–5.0) within the cells. The dissociation of DCA-Pt(II) results in an aquated cisplatin and two DCA molecules. They work in a concerted manner: (i) the aquated cisplatin blocks the DNA replica and induces cell apoptosis, and (ii) the released DCA depolarizes the cell mitochondrion through inhibition of glycolysis and decrease of Δψm, inducing irreversible apoptosis (Scheme 1). In this manner, we expect that some synergy will arise.
3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was purchased from Sigma-Aldrich (St Louis, Missouri, USA). All other chemicals and solvents were used as received.
Synthesis of DCA-Pt(II)
In brief, cisplatin (0.30 g, 1 mmol), HNO3 (100 μl), and silver nitrate (0.34 g, 2 mmol) were added into a roundbottom flask containing 50 ml pure water. The reaction mixture was stirred in the dark at room temperature for 48 h. The resultant turbid solution was filtered to remove white silver chloride. To the yellow colored solution, sodium dichloroacetate (0.36 g, 2.4 mmol) was added to form a precipitate of DCA-Pt(II). The solid was filtered
Materials and methods Materials
Cisplatin (purity 99%) was purchased from Shandong Boyuan Pharmaceutical Co. Ltd (Jinan, Shandong, China). Scheme 1
(a)
O
CI
O
CI
O CI
O
CI
H3N
H3N
O
H3N
Pt
Pt
Pt CI
H3N
CI
HO
Cisplatin
O
O
H3N
H3N
O
O Carboplatin
DCA
CI CI
DCA-Pt(II)
pH=7.4, [CI−]= 100 mmol/l
(b)
pH=5.0, [CI−]= 4−10 mmol/l
II)
t( A-P
DC
ed
n
atio
Aqu
DCA releas
Mitochondrion
CI H 2O Pt
H3N
O
A
d
se
ea
rel
DC
Depolarization
tion
Aqua
H 3N
CI
O
NH3 H2O H3N
Pt
Pt NH3
H2O DNA binding
H3N
g
kin
slin
ros
C
Cancer cells Possible mechanism of action of DCA-Pt(II) in cancer cells. (a) Cisplatin, DCA, carboplatin, and DCA-Pt(II); (b) DCA-Pt(II), passively diffusing into the cancer cells, undergoes aquation, facilitating the release of two DCA molecules and aquated cisplatin. Thereafter, DCA depolarizes the mitochondrion, whereas cisplatin crosslinks with DNA. The concerted action reverses cisplatin resistance. DCA, dichloroacetate; Pt, platinum.
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700 Anti-Cancer Drugs 2015, Vol 26 No 7
and dried under vacuum. A weight of 0.42 g of the product was obtained in an 87% yield. Cell lines and culture conditions
A2780 and A2780DDP cells were purchased from the Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, Shanghai, China, and grown in RPMI-1640 medium (Life Technologies) supplemented with 10% fetal bovine serum, 0.03% L-glutamine, and 1% penicillin/streptomycin at 37°C in a 5% CO2 atmosphere. LO2 human liver cells were purchased from the American Type Culture Collection and cultured in RPMI-1640 medium supplemented with 10% newborn calf serum, 0.03% L-glutamine, and 1% penicillin/streptomycin at 37°C in a 5% CO2 atmosphere. MTT assay
A2780 and A2780DDP cells harvested in the logarithmic growth phase were seeded in 96-well plates at a density of 1 × 104 cells/well and incubated with RPMI-1640 medium for 12 h. The medium was then replaced with RPMI-1640 medium containing either (i) cisplatin, cisplatin + 2DCA, and DCA-Pt(II) at a final equivalent Pt concentration of 0.2–108 μmol/l, (ii) carboplatin and carboplatin + 2DCA at a final equivalent Pt concentration of 0.2–432 μmol/l, or (iii) DCA at a two concentration ranges (0–200 μmol/l and 312.5–50 mmol/l), which was used as a control. The incubation time for all drugs was 48 h. After incubation, 20 μl of MTT solution in PBS with a concentration of 5 mg/ml was added to the wells, and the plates were incubated for another 4 h at 37°C, followed by removal of the culture medium containing MTT and addition of 150 μl dimethyl sulfoxide to each well to dissolve the formazan crystals formed. Finally, the plates were shaken for 10 min, and the absorbance of the formazan product was measured at 492 nm on a microplate reader. Determination of Pt content in the cancer cells
A2780 and A2780DDP were seeded in six-well plates at a density of 1 × 106 cells/well. At their logarithmic phase of growth, the cells were treated with cisplatin, cisplatin + 2DCA, DCA-Pt(II), carboplatin, and carboplatin + 2DCA, with final Pt concentrations in the culture medium regulated to 10 μmol/l, and incubated at 37°C for 1 and 4 h, respectively. As described previously [14,16,17], to remove surface-bound drugs the cells were washed three times with ice-cold PBS, incubated with 1.5 ml of 0.15 mol/l sodium chloride (pH 3.0 was adjusted by acetic acid) for 3 min at 4°C, rinsed with 2 ml of cold PBS, harvested by scraping in ice-cold PBS, and centrifuged. Thereafter, the cell pellets were lysed by adding 200 μl of cell lysis buffer (Promega, Beijing, China), and the cell lysis solution was frozen at − 20°C for 20 min and thawed at room temperature. For each sample, 100 μl of the cell lysis solution was used to directly measure the Pt content by inductively coupled plasma mass spectrometry (ICPMS). The other 100 μl of the cell lysis solution was used
to determine the protein content in each cell sample using the bicinchoninic acid protein assay kit (Beyotime Institute of Biotechnology, Shanghai, China) according to previously published data [14,16,17]. The Pt content was expressed as nanograms of Pt per milligram of total proteins. Determination of Pt-DNA adduct contents in the cancer cells
A2780 and A2780DDP were seeded in six-well plates at a density of 1 × 106 cells/well. At their logarithmic phase of growth, the cells were treated with cisplatin, cisplatin + 2DCA, DCA-Pt(II), carboplatin, and carboplatin + 2DCA, with the final Pt concentration in the culture medium regulated to 2 μmol/l, and incubated at 37°C for 24 h. Next, we used a genomic DNA extraction kit (Tiangen Biotech, Beijing, China) to isolate genomic DNA from the cells treated with the different drugs, and we finally added 200 μl distilled water to dissolve the DNA. Thereafter, we used Nano drop to measure the concentration of DNA with 1 μl DNA. The remaining 200 μl of DNA was diluted to 1 ml in volume. The Pt content was measured by ICP-MS and expressed as nanograms of Pt per milligram of total genomic DNA. MMP analysis
It is well known that MMP (Δψm) is an important parameter in the mitochondrial function of cells. JC-1 (Beyotime Biotech; 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethyl-imidacarbocyanine iodide) is a cationic and lipophilic dye that can enter the mitochondria and change its fluorescence color on the basis of a change in the MMP (Δψm), as it exists in the monomeric form at low Δψm, at which it shows green fluorescence (∼520 nm), and it exists as ‘Jaggregates’ at high Δψm, which fluoresce red (∼590 nm). Therefore, it is often used to characterize MMP. After the cells are stained with JC-1, their green fluorescence and red fluorescence are detected, respectively, and the intensity ratio of green/red fluorescence is considered as a measure of Δψm of the cells. On the basis of the green/red ratio, mitochondrial function or cellular function can be deduced. In the present study, A2780 and A2780DDP cells were seeded into black 96-well microplates. After 24 h, the drugs [cisplatin, cisplatin + 2DCA, DCA-Pt(II), carboplatin, and carboplatin + 2DCA at 50 μmol/l Pt and 100 μmol/l DCA concentrations] were added to the wells for 1 h incubation, and the cells without treatment were used as controls. At the end of drug treatment, the medium was removed and the cells were incubated at 37°C for 1 h with 5 mg/l of JC-1. Thereafter, they were washed twice with PBS and placed in fresh medium without serum. Following this, images were viewed by high-throughput fluorescence confocal microscopy (BD Pathway Bioimager 855; BD Biosciences, San Diego, California, USA) at an excitation wavelength of 488 nm and an emission wavelength of 530 nm for green, and at an excitation wavelength of 540 nm and an emission wavelength of 590 nm for red fluorescence. The intensities
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DCA-Pt(II) overcomes cisplatin drug resistance Zhang et al. 701
Fig. 1
(a)
(b)
100
500 A2780
A2780
431.85
A2780DDP
404.75
400
A2780DDP
IC50 values (μmol/l)
60
40
20
338.5 317.4
300 200 84.5
100
62.5
22.1
18.9
21.35 31.25
pl at
ar
in +2 D
bo pl at
C
A
in
A
bo ar C
C
isp
C
D
C
A-
in +2 D
C
Pt (I
in la t
200
isp
100 Pt concentration (μmol/l)
C
0
I)
0
0
la t
Cell viability (%)
80
Effects of various drugs on cisplatin-sensitive ovarian cancer cells, A2780, and cisplatin-resistant cells, A2780DDP (mean ± SD, n = 3). (a) Representative dose-dependent cell viability curve of cisplatin and (b) IC50 of various drugs. Data above the columns are the IC50 values based on Pt read. For the tested drugs cisplatin + 2DCA and carboplatin + 2DCA, the molar ratio of DCA to Pt is 2 : 1. DCA alone at a concentration less than 10 mmol/l shows no difference in the inhibition of A2780 and A2780DDP cancer cell lines (IC50 of DCA is 33 and > 50 mmol/l in A2780 and A2780DDP, respectively; Fig. S2, Supplemental digital content 1, http://links.lww.com/ACD/A104). DCA, dichloroacetate; IC, inhibitory concentration; Pt, platinum.
of both green fluorescence (JC-1 monomers) and red fluorescence (J-aggregates) were measured using a FLUOstar optima plate reader (BMG Labtech, Offenburg, Germany), with the filters set to 485 nm excitation/520 nm emission (green) and 544 nm excitation/590 nm emission (red), and the data were presented as the ratio of green to red signals (520/590 nm), which was normalized to that for the nontreated cells.
Western blot of cleaved-caspase-3 and cleaved-caspase-9
A2780 and A2780DDP were seeded in six-well plates at a density of 1 × 106 cells/well. At their logarithmic phase of growth, the cells were treated with drugs [cisplatin, cisplatin + 2DCA, DCA-Pt(II), carboplatin, and carboplatin + 2DCA at 50 μmol/l Pt and 100 μmol/l DCA concentrations] and incubated at 37°C for 10 h. Thereafter, the cells were harvested and rinsed twice with PBS. Cell extracts were treated with lysis buffer [1% Nonidet P-40, 50 mmol/l Tris-HCl (pH 7.5), 150 mmol/l NaCl, 1 mmol/l NaF, 1 mmol/l phenylmethylsulfonyl fluoride, 4 μg/ml leupeptin, and 1 μg/ml aprotinin] for 30 min with occasional rocking, followed by centrifugation at 15 000 rpm for 15 min at 4°C. Identical amounts (100 μg of protein) of cell lysate were resolved by 10% SDS-PAGE, transferred onto a PVDF membrane, and then blocked with 5% fat-free dry milk in TBST [20 mmol/l Tris-HCl (pH 7.6), 150 mmol/l NaCl, and 0.02% Tween-20] for 1 h at room temperature. The
membrane was immunoblotted with rabbit anti-human cleaved-caspase-3 and cleaved-caspase-9 polyclonal antibodies (1 : 1000; Cell Signaling, Boston, Massachussetts, USA) in 1% milk/TBST. To assure equivalent protein loading, the membranes were simultaneously incubated with GAPDH monoclonal antibodies (1 : 1000; Kangcheng Co., Shanghai, China) at 4°C overnight. The membranes were then probed with diluted primary antibodies in 1% milk/TBST for 24 h at 4°C, washed three times, incubated with horseradish peroxidase-conjugated secondary antibodies for 30 min at room temperature, and washed extensively before detection by chemiluminescence using the ECL-Plus kit (Beyotime Biotech). Proteins were visualized by exposing the blots to Kodak film (Eastman Kodak, Rochester, New York, USA). Western blotting data were quantified using Image J software (National Institutes of Health, Bethesda, Maryland, USA).
Cell cycle analysis
Flow cytometry was used to evaluate the number of cells in specific phases of the cell cycle. Cell cycle distribution was determined by staining DNA with propidium iodide. A2780 and A2780DDP were seeded in six-well plates at a density of 1 × 106 cells/well. At their logarithmic phase of growth, drugs [cisplatin, cisplatin + 2DCA, and DCA-Pt (II) at 20 μmol/l Pt concentration] were added to the wells and the cells were incubated for 16 h; cells without treatment were used as controls. After treatment, cells were collected, washed twice with ice-cold PBS buffer
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702 Anti-Cancer Drugs 2015, Vol 26 No 7
Fig. 2
Intracelluar uptake (ng Pt/mg protein)
(a)
300 250
1 h A2780
1 h A2780DDP
4 h A2780
4 h A2780DDP
200 150 100 50
C
A
in
A
C
C
isp
C
ar bo pl at
in +2 D
ar bo pl at
C in +2 D la t
D
C
C
A-
isp
la t
Pt (I
in
I)
0
200 150 A2780DDP
A2780 100 50
C
A
in
in +2 D
pl bo
at
ar
pl
C
bo ar C
C
isp
la
D
tin
C
A-
+2 D
at
C
(II Pt
la isp C
A
)
0 tin
Pt-DNA adducts (pg Pt/μg protein)
(b)
Intracellular uptake and Pt-DNA adducts (mean ± SD, n = 3). (a) Drug uptake by A2780 and A2780DDP cells over 1 and 4 h. (b) Pt-DNA adducts of drugs in A2780 and A2780DDP cells incubated for 24 h. DCA, dichloroacetate; Pt, platinum.
(pH 7.4), fixed with 70% alcohol at 4°C overnight, and then stained with propidium iodide (1 mg/ml) in the presence of 1% RNAase A at 37°C for 30 min. The percentages of cells in different phases of the cell cycle were measured using a flow cytometer (Beckman Coulter Epics; Beckman Coulter, Pasadena, California, USA), and the percentages of cells in G0/G1, S, and G2/M phases were analyzed using MultiCycle software (Microsemi Corp., Aliso Viejo, California, USA). Statistical analysis
The data were expressed as mean ± SD. Student’s t-test was used to determine the statistical difference between various experimental and control groups. Differences were considered statistically significant at P less than 0.05.
Results and discussion Cell viability studies
We show a representative dose-dependent cell viability curve of cisplatin for the epithelial ovarian cancer cell line A2780 and its acquired resistant cell line A2780DDP (Fig. 1a). A2780DDP shows a much more inert response to cisplatin, and the cell viability was more than 20% even at the highest concentration of 200 µmol/l, indicating the inability of cisplatin to kill the cancer cells, which
may be the major cause of clinical tumor recurrence. The IC50 of cisplatin for A2780 and A2780DDP were 22.1 and 84.5 µmol/l, respectively, indicating a resistance of ∼ 3.8-fold (Fig. 1b). DCA does not markedly inhibit the two cell lines even at a concentration of up to 10 mmol/l (Fig. S2, Supplemental digital content 1, http:// links.lww.com/ACD/A104). To test the efficacy of DCA-Pt (II) in reversing drug resistance, a mixture of cisplatin and two molar equivalent DCA (cisplatin + 2DCA) and a mixture of carboplatin and carboplatin with two molar equivalent DCA (carboplatin + 2DCA) were chosen as controls. DCA-Pt(II) is found to be the most effective drug among all the tested groups in the two cancer cell lines. Its IC50 values were 18.9 and 21.35 µmol/l for A2780 and A2780DDP, respectively, implying a resistance of ∼ 1.1-fold, which is markedly lower than that of cisplatin (∼3.8-fold). Therefore, DCA-Pt(II) reversed cisplatin drug resistance by 3.5-fold. DCA-Pt(II) has more similarity to carboplatin in chemical structure as both share moderately liable leaving groups, that is, carboxylic acids, whereas cisplatin has two chloride ions as readily liable leaving groups. Therefore, to some extent, it makes more sense to compare DCA-Pt(II) with carboplatin and carboplatin + 2DCA. We found that DCA-Pt(II) is ∼ 18 and 19 times more effective than carboplatin, and 16.8 and 20.2 times more effective than carboplatin + 2DCA in A2780 and A2780DDP, respectively. The results indicate that DCA-Pt(II) is the most effective drug in both ovarian cancer cell lines and can help overcome drug resistance in ovarian cancer. An ideal anticancer drug should be less toxic to healthy cells. Therefore, we analyzed the toxicity of these drugs in normal liver cells (L02) over 6–24 h, which showed that DCA-Pt(II) has a lower toxicity than cisplatin and cisplatin + 2DCA under all conditions (Fig. S1, Supplemental digital content 1, http://links.lww.com/ACD/A104). Pt and Pt-DNA adduct content test in cells
Pt(II)-based drugs are believed to passively diffuse into cancer cells [18] and bind to cellular biomolecules (amino acid, proteins, RNA, DNA, etc.) after their subsequent intracellular dissociation. For cisplatin and carboplatin, it is generally believed that the efficacy of Pt(II) drugs is great determined by the amount of intracellular drug and the ultimate Pt-DNA adducts [14]. However, compared with A2780 cells, the difference in biological characteristics (lower levels of membrane protein copper transporter 1, higher levels of intracellular glutathione and DNA repair genes and proteins) of resistant A2780DDP cells makes them internalize fewer Pt drugs, and hence, fewer Pt-DNA adducts are formed [4–6]. To provide insight into the higher efficacy and possible reason for overcoming drug resistance of DCA-Pt(II), we first determined the intracelluar uptake of various drugs [cisplatin, DCA-Pt(II), cisplatin + 2DCA, carboplatin, and carboplatin + 2DCA] by A2780 and A2780DDP
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DCA-Pt(II) overcomes cisplatin drug resistance Zhang et al. 703
1
GAPDH
0.5
A
I) ar b C op ar la bo tin pl at in +2 D C A
C 2D
A2780/DDP
I)
Pt (I
C
C
A-
la tin
C
C
isP t(I
C
Procaspase-9 Cleaved-caspase-9 Cleaved-caspase-3
C
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D
isp
C
la t
C
A-
isp
in +2 D
la t
A
in
0 on t
isp
on tro l C isp la tin C isp la tin D + C A- 2DC Pt A (II C ) ar bo pl at C in ar bo pl 2D at in C +2 A D C
(c)
C
isp
A
1.5
Procaspase-9 Cleaved-caspase-9 Cleaved-caspase-3
D
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on t
A2780 2
C
A2780DDP
ro l
A2780
C
2.5
la tin
+2 D
C
(b)
ro l
Mitochondrial membrane potential (green/red)
(a)
A ar bo pl at C in ar bo p la 2D tin C +2 A D C
A
Fig. 3
GAPDH (e)
A C
at
2D
A D
C
in
bo ar
pl
C
in +2
pl
Pt
at
(II
A
AC
la
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ar
bo
isp C
bo ar C
C
tro
A C
pl
C
)
0
at
2D
A C
in +2
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at pl
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+2 tin
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isp
la
in
) (II
A C D
la isp C
C
on
tro
l
tin
0
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D
50
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D
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tin
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tin +2
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l
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isp
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C
A2780DDP
on
A2780
C
400
Cleaved caspase-9/GAPDH
Cleaved caspase-3/GAPDH
(d)
DCA-Pt(II) induced mitochondria-mediated apoptosis in A2780 and A2780DDP cells. (a) The mitochondrial potential changes as revealed by the JC-1 assay; (b) A2780 cell lines; (c) A2780/DDP cell lines; (d) quantitative analysis of cleaved-caspase-3; (e) cleaved-caspase-9 of the two cell lines treated with various drugs. DCA, dichloroacetate; Pt, platinum; JC-1, 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethyl-imidacarbocyanine iodide.
during 1 and 4 h of drug incubation using ICP-MS. The results presented in Fig. 2a indicate that (i) the uptake of cisplatin by both A2780 and A2780DDP is time dependent. From 1 to 4 h, more drugs were taken up. Drug accumulation by drug-resistant A2780DDP cells is 48% of that by A2780 cells at 1 h (9.1 vs. 18.9 ng Pt/mg protein) and 42% at 4 h (99.4 vs. 239.1 ng Pt/mg protein); (ii) adding DCA to cisplatin/carboplatin to make a physical mixture seems to reduce the drug uptake (cisplatin vs. cisplatin + 2DCA; carboplatin vs. carboplatin + 2DCA), whereas fusion of DCA and cisplatin to one molecule of DCA-Pt(II) was also shown to reduce the drug uptake by both cell lines at 1 and 4 h. (iii) At 1 and 4 h, DCA-Pt(II) leads to the accumulation of the least amount of drugs in both cell lines, which is seemingly contradictory to the
fact that DCA-Pt(II) is the most effective and overcomes cisplatin resistance. Next, we tested the Pt-DNA adducts in the two cell lines after 24 h of drug incubation at an equal Pt concentration of 2 µmol/l (Fig. 2b). Consistent results can be found, as follows: (i) fewer Pt-DNA adducts were found in A2780DDP compared with A2780 cells for all the drugs; (ii) cells treated with cisplatin or cisplatin + 2DCA form many more Pt-DNA adducts than those treated with DCA-Pt(II), carboplatin, or carboplatin + 2DCA. This is due to the higher inertness of the carboxylic leaving group of carboplatin and DCA-Pt (II), whereas cisplatin has more liable chloride leaving groups; (iii) apart from drug groups with carboplatin, DCA-Pt(II) forms the fewest Pt-DNA adducts. Considering the fact that DCA-Pt(II) can overcome
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704 Anti-Cancer Drugs 2015, Vol 26 No 7
Table 1
Proportion of A2780 and A2780DDP cells in each phase of the cell cycle G0/G1 phase
Control Cisplatin Cisplatin + 2DCA DCA-Pt(II)
S phase
G2/M phase
A2780 (%)
A2780DDP (%)
A2780 (%)
A2780DDP (%)
A2780 (%)
A2780DDP (%)
66.85 ± 0.85 30.90 ± 0.65 31.49 ± 1.02 42.92 ± 1.33
55.48 ± 0.54 49.90 ± 0.90 50.60 ± 1.12 49.17 ± 1.36
22.11 ± 0.47 60.96 ± 0.32 63.26 ± 2.15 57.08 ± 1.89
28.66 ± 0.38 36.83 ± 0.85 36.87 ± 1.23 46.58 ± 1.40
11.05 ± 0.23 8.14 ± 0.58 5.24 ± 0.13 0.00 ± 0.12
15.86 ± 0.23 13.27 ± 0.47 12.53 ± 0.78 4.25 ± 0.12
Results represent the mean ± SD of three independent experiments. DCA, dichloroacetate; Pt, platinum.
cisplatin resistance with highest efficacy, the lower amount of Pt intracellular uptake and Pt-DNA adducts indicate other possible cellular pathways, unlike those involved with cisplatin, to kill more cancer cells.
Mitochondrion-dependent apoptosis research
DCA-Pt(II) has two DCA molecules; hence, it may exert some effect on the mitochondria. To find the possible mechanism for overcoming drug resistance, the cell mitochondrial transmembrane potential was measured using the established JC-1 assay [15]. JC-1 can selectively enter the mitochondria and reversibly change color from red to green as the membrane potential reduces. In healthy cells with high mitochondrial transmembrane potential, JC-1 forms complexes known as J-aggregates, which fluoresce intense red. In apoptotic cells with low mitochondrial transmembrane potential, JC-1 remains in the monomeric form, which fluoresces green. Therefore, a greater green/red ratio in fluorescence indicates more cell death. Figure 3a shows the green to red signals of cells treated with various drugs. DCA has an effect on the change in the red to green fluorescence ratio, indicating a high ability for reducing the mitochondrial membrane potential and inducing apoptosis. However, cisplatin and carboplatin lead to little alteration in the green to red ratio, suggesting limited effects on depolarization of the mitochondrion and inducing its dysfunction. Adding DCA to cisplatin (cisplatin + 2DCA) or carboplatin (carboplatin + 2DCA) resulted in a great change in the green/red fluorescence ratio, whereas DCA-Pt(II) seems to have the largest effect on depolarization of the mitochondrion and its dysfunction in both cell lines. We also found that DCA-Pt(II) had an even larger influence on the resistance A2780DDP compared with A2780. The results here indicate that DCA-Pt(II) has a great effect on the mitochondria, which mediates the cellular apoptotic pathway. This differs from the present Pt drugs and is possibly the reason that DCA-Pt(II) is much more effective than other drugs in the two cell lines and that DCA overcomes cisplatin drug resistance. In mitochondrion-dependent apoptosis, exposure of cells to various stress signals triggers the disruption of the mitochondrion, leading to the release of cytochrome c into the cytosol [19]. The apoptosomes formed contain cytochrome c, Apaf-1, and caspase-9. The activation of
caspase-9 leads to the cleavage and activation of executioner caspase-3, leading to apoptosis [20]. Hence, we test the activation of caspase-9 and caspase-3 in A2780 and A2780DDP cells treated with different drugs. As shown in Fig. 3b and c, cisplatin can induce activation of caspase-3 to a larger extent in A2780 cells than in A2780DDP cells. In the presence of DCA, the effects of cisplatin in A2780DDP cells seems better. However, DCA-Pt(II) had the largest effect on caspase-3 in both A2780 parental cells and A2780DDP resistant cells. We can also see a clear difference in cleaved-caspase-3 levels between A2780 and A2780DDP cells in quantitative analysis (Fig. 3d), further suggesting drug resistance of A2780DDP cells to cisplatin. As caspase-9 acts upstream of caspase-3 and activates caspase-3 leading to apoptosis, we measure the activation of caspase-9 induced by Pt drugs. We found that adding DCA to cisplatin (cisplatin + 2DCA) and carboplatin (carboplatin + 2DCA) both resulted in the activation of caspase-9 in A2780DDP cells, whereas DCA-Pt(II) induced more obvious activation of caspase-9 in both A2780 cells and A2780DDP cells (Fig. 3e). We also found that DCA can induce more activation of caspase-9, which makes A2780DDP cells more sensitive to cisplatin, but has no effect on the activation of caspase-3. Therefore, these data above suggest that DCA-Pt(II) is much more effective than other drugs especially in A2780DDP cells, which is possibly related to caspase-9-mediated mitochondrial dysfunction, which further leads to cell apoptosis.
Effect of platinum drugs on cell cycle distribution
The cell cycle is typically divided into different phases: quiescent and gap 1 (G0/G1 phase), synthesis (S phase), and gap 2 and mitosis (G2/M phase), which governs the transition from quiescence through cell growth to proliferation. To determine whether the effects of Pt drugs on proliferation are mediated by inhibition of cell cycle progression, the cell cycle phase distribution was analyzed in A2780 and A2780DDP cells by flow cytometry. Cells were treated with drugs [cisplatin, cisplatin + 2DCA, DCA-Pt(II) at a Pt concentration of 20 μmol/l] for 16 h. As shown in Table 1, incubation of A2780 cells leads to redistribution of the percentage of cells in the phases of the cell cycle. Cisplatin alone arrested A2780 and
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DCA-Pt(II) overcomes cisplatin drug resistance Zhang et al. 705
A2780DDP cells at the S phase. When the cells were treated with ‘cisplatin + 2DCA’, the percentage of both cell lines in the G2/M phase decreased by 6 and 3%, respectively. The results indicate that DCA can promote cell cycle arrest induced by cisplatin. In particular, no A2780 cells stayed in the G2/M phase after DCA-Pt(II) treatment, and only 4.25% of A2780DDP cells stayed in the G2/M phase with the same treatment. A 35% increase in A2780 cells in the S phase and a 11% decrease in A2780 cells in the G2/M phase were seen on treatment with DCA-Pt(II). At the same time, there was an 18% increase in A2780DDP cells in the S phase and an ∼ 11% decrease in A2780DDP cells in the G2/M phase after treatment with DCA-Pt(II). These results suggest that the DCA-Pt(II) blocked cell cycle progression more effectively than other drugs. Conclusion
References 1 2 3
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5 6
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Here, we found that DCA-Pt(II), which is different from clinical Pt drugs used currently, overcomes ovarian cancer drug resistance by dual organelle targeting. Despite less cellular uptake and fewer Pt-DNA adducts present, DCAPt(II) induces much more mitochondrial dysfunction, as well as caspase-3 and caspase-9 cleavage, thereby resulting in greater levels of apoptosis in both sensitive and resistant cell lines. Furthermore, DCA-Pt(II) induced S phase arrest in A2780 parental cells and A2780DDP resistant cells. Although the detailed mechanism of action requires further investigation, the concerted manner of targeting different organelles makes DCA-Pt(II) a candidate for further study, especially in drug-resistant cancer.
Acknowledgements This work is supported by the National Natural Science Foundation of China (Nos. 51403031, 31170324, and 31070318), the China Postdoctoral Science Foundation (No. 2014M550163), the Research Funds from Science & Technology Department of Jilin Province (Nos. 20140520049JH, and 20130201008ZY), and the funds from the Science & Technology Department of Changchun City (No. 2012098).
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Conflicts of interest
There are no conflicts of interest.
Tagawa T, Morgan R, Yen Y, Mortimer J. Ovarian cancer: opportunity for targeted therapy. J Oncol 2012; 2012:682480. Zeineldin R, Muller CY, Stack MS, Hudson LG. Targeting the EGF receptor for ovarian cancer therapy. J Oncol 2010; 2010:414676. Xiao H, Qi R, Liu S, Hu X, Duan T, Zheng Y, et al. Biodegradable polymer – cisplatin(IV) conjugate as a pro-drug of cisplatin(II). Biomaterials 2011; 32:7732–7739. Parker RJ, Eastman A, Bostick-Bruton F, Reed E. Acquired cisplatin resistance in human ovarian cancer cells is associated with enhanced repair of cisplatin-DNA lesions and reduced drug accumulation. J Clin Invest 1991; 87:772–777. Boulikas T, Vougiouka M. Cisplatin and platinum drugs at the molecular level (Review). Oncol Rep 2003; 10:1663–1682. Kikuchi Y, Iwano I, Miyauchi M, Kita T, Oomori K, Kizawa I, et al. The mechanism of acquired resistance to cisplatin by a human ovarian cancer cell line. Jpn J Cancer Res 1988; 79:632–635. Yu Z, Schmaltz RM, Bozeman TC, Paul R, Rishel MJ, Tsosie KS, Hecht SM. Selective tumor cell targeting by the disaccharide moiety of bleomycin. J Am Chem Soc 2013; 135:2883–2886. Bhattacharya C, Yu Z, Rishel MJ, Hecht SM. The carbamoylmannose moiety of bleomycin mediates selective tumor cell targeting. Biochemistry 2014; 53:3264–3266. Gottlieb E, Armour SM, Harris MH, Thompson CB. Mitochondrial membrane potential regulates matrix configuration and cytochrome c release during apoptosis. Cell Death Differ 2003; 10:709–717. Kroemer G, Galluzzi L, Brenner C. Mitochondrial membrane permeabilization in cell death. Physiol Rev 2007; 87:99–163. Yu Z, Li J, Zhu J, Zhu M, Jiang F, Zhang J, et al. A synthetic fourth transmembrane segment derived from TRPV4 channel self-assembles into a potassium channel to regulate the vascular smooth muscle cell membrane potential. J Mater Chem B 2014; 2:3809–3818. Michelakis ED, Webster L, Mackey JR. Dichloroacetate (DCA) as a potential metabolic-targeting therapy for cancer. Br J Cancer 2008; 99:989–994. Xiao L, Li X, Niu N, Qian J, Xie G, Wang Y. Dichloroacetate (DCA) enhances tumor cell death in combination with oncolytic adenovirus armed with MDA-7/IL-24. Mol Cell Biochem 2010; 340:31–40. Xiao H, Yan L, Zhang Y, Qi R, Li W, Wang R, et al. A dual-targeting hybrid platinum(IV) prodrug for enhancing efficacy. Chem Commun (Camb) 2012; 48:10730–10732. Song H, Xiao H, Zhang Y, Cai H, Wang R, Zheng Y, et al. Multifunctional Pt (IV) pro-drug and its micellar platform: to kill two birds with one stone. J Mater Chem B 2013; 1:762–772. Xiao H, Zhou D, Liu S, Zheng Y, Huang Y, Jing X. A complex of cyclohexane1,2-diaminoplatinum with an amphiphilic biodegradable polymer with pendant carboxyl groups. Acta Biomater 2012; 8:1859–1868. Xiao H, Song H, Zhang Y, Qi R, Wang R, Xie Z, et al. The use of polymeric platinum(IV) prodrugs to deliver multinuclear platinum(II) drugs with reduced systemic toxicity and enhanced antitumor efficacy. Biomaterials 2012; 33:8657–8669. Wang D, Lippard SJ. Cellular processing of platinum anticancer drugs. Nat Rev Drug Discov 2005; 4:307–320. Mahoney S, Arfuso F, Rogers P, Hisheh S, Brown D, Millward M, Dharmarajan A. Cytotoxic effects of the novel isoflavone, phenoxodiol, on prostate cancer cell lines. J Biosci 2012; 37:73–84. Chen M, Guerrero AD, Huang L, Shabier Z, Pan M, Tan TH, Wang J. Caspase-9-induced mitochondrial disruption through cleavage of antiapoptotic BCL-2 family members. J Biol Chem 2007; 282: 33888–33895.
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Preclinical report
A hybrid platinum drug dichloroacetate-platinum(II) overcomes cisplatin drug resistance through dual organelle targeting Yu Zhanga,*, Guannan Guob,*, Ben Maa, Rong Dua, Haihua Xiaoc, Xiaoguang Yangb, Wenliang Lia, Ying Gaod, Yuxin Lib and Xiabin Jingc A hybrid drug dichloroacetate-platinum(II) [DCA-Pt(II)] was found to overcome cisplatin drug resistance of ovarian cancer through a dual targeting mode, which is different from the mode of action of the present platinum (Pt) drugs used in clinics. DCA-Pt(II) exhibited remarkable cytotoxicity against both cisplatin-sensitive (A2780) and cisplatinresistant (A2780DDP) ovarian cancer cells. The Pt and Pt-DNA adduct content test showed that there was less Pt cellular uptake and fewer Pt-DNA adducts were present after DCA-Pt(II) treatment compared with treatment with cisplatin, carboplatin, and some other drugs. In the study, the effects of DCA-Pt(II) on the cell cycle and apoptosis were also investigated, which showed that DCA-Pt(II) induced G2/M phase arrest and mitochondria-mediated apoptosis in both sensitive and resistant cells lines. Interestingly, DCA-Pt(II) had much greater effects on mitochondria in A2780DDP cell lines than in A2780 cell lines. Anti-Cancer Drugs 26:698–705 Copyright © 2015 Wolters Kluwer Health, Inc. All rights reserved.
Introduction Ovarian cancer is the most common cause of gynecologic neoplasms and the fifth most common cause of cancer mortality among women worldwide [1]. The mortality rate among women with ovarian cancer, especially for epithelial ovarian cancer (85% of all cases of ovarian cancer), is quite high [1,2]. Despite marked progress in surgical techniques, as well as in treatment options, the 5-year survival rate for stage III/IV ovarian cancer still remains at ∼ 45% [1,2]. Cisplatin is a DNA alkylating agent [3,4] and has been the most active drug for ovarian cancer for decades [4,5]. However, although most women with ovarian cancer respond to frontline platinum (Pt) drugs, the majority of them develop tumors resistant to Pt drugs [5,6]. Resistance to Pt drugs falls into two major groups: (i) limited Pt-DNA adduct formation (reduced drug internalization and enhanced detoxification through thiols, proteins, etc.) and (ii) cell signaling pathway alteration, which prevents cell death from formation of Pt-DNA adducts (increased Pt-DNA damage tolerance or enhanced repair of damaged DNA) [4–6].
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.
Anti-Cancer Drugs 2015, 26:698–705 Keywords: antitumor, dichloroacetate, drug resistance, organelle targeting, platinum drug a
National Engineering Laboratory for Druggable Gene and Protein Screening, Jilin Province Key Laboratory on Chemistry and Biology of Natural Drugs in Changbai Mountain, School of Life Sciences, Northeast Normal University, cState Key Laboratory of Polymer Physics and Chemistry, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences and dSchool of Chemistry & Environmental Engineering, Changchun University of Science and Technology, Changchun, China b
Correspondence to Wenliang Li, PhD, National Engineering Laboratory for Druggable Gene and Protein Screening, Northeast Normal University, Changchun 130024, China Tel: + 86 431 89165937; fax: + 86 431 89165917; e-mail: [email protected] and Yuxin Li, PhD, Jilin Province Key Laboratory on Chemistry and Biology of Natural Drugs in Changbai Mountain, School of Life Sciences, Northeast Normal University, Changchun 130024, China Tel/fax:+ 86 431 89165926; e-mail: [email protected] *Yu Zhang and Guannan Guo contributed equally to the writing of this article. Received 31 October 2014 Revised form accepted 24 February 2015
Cell targeting is the key to cancer treatment [7,8]. The mitochondrion is the power plant of cells, providing energy for various cellular activities. Tumor cells have altered metabolism through conversion of glucose oxidation to glycolysis. The excessive glycolysis results in hypoxia, evading apoptosis and leading to unlimited proliferation. Moreover, cancer cells have a hyperpolarized mitochondrial membrane potential (MMP, Δψm). Loss of Δψm is an early event in apoptotic cascades [9–11]. Therefore, the mitochondria might be a potential cancer target. Dichloroacetate (DCA) can enhance apoptosis by increasing the movement of pyruvate into the mitochondria, shifting the metabolism from glycolysis to glucose oxidation and decreasing Δψm. This eventually leads to an increase in apoptosis and a decrease in cellular proliferation [12,13]. Thus, DCA causes cancer regression by sensitizing cells to chemotherapy through the mitochondrial apoptotic pathway, with minimal effects on normal cells [14]. A series of Pt drugs were developed by our group [14,15]. Among them, a hybrid Pt(II) drug, DCA-Pt(II), with both DCA and Pt in one molecule [15] showed a concerted mode of action and good efficacy in tumor inhibition. We envisage here that DCA-Pt(II) can overcome cisplatin drug resistance through dual targeting organelles. This drug can undergo subsequent dissociation and hydration after DOI: 10.1097/CAD.0000000000000234
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DCA-Pt(II) overcomes cisplatin drug resistance Zhang et al. 699
internalization by the cancer cells because of the lower chloride concentrations (from ∼ 100 to 2–10 mmol/l) and lower pH values (pH = 7.4–5.0) within the cells. The dissociation of DCA-Pt(II) results in an aquated cisplatin and two DCA molecules. They work in a concerted manner: (i) the aquated cisplatin blocks the DNA replica and induces cell apoptosis, and (ii) the released DCA depolarizes the cell mitochondrion through inhibition of glycolysis and decrease of Δψm, inducing irreversible apoptosis (Scheme 1). In this manner, we expect that some synergy will arise.
3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was purchased from Sigma-Aldrich (St Louis, Missouri, USA). All other chemicals and solvents were used as received.
Synthesis of DCA-Pt(II)
In brief, cisplatin (0.30 g, 1 mmol), HNO3 (100 μl), and silver nitrate (0.34 g, 2 mmol) were added into a roundbottom flask containing 50 ml pure water. The reaction mixture was stirred in the dark at room temperature for 48 h. The resultant turbid solution was filtered to remove white silver chloride. To the yellow colored solution, sodium dichloroacetate (0.36 g, 2.4 mmol) was added to form a precipitate of DCA-Pt(II). The solid was filtered
Materials and methods Materials
Cisplatin (purity 99%) was purchased from Shandong Boyuan Pharmaceutical Co. Ltd (Jinan, Shandong, China). Scheme 1
(a)
O
CI
O
CI
O CI
O
CI
H3N
H3N
O
H3N
Pt
Pt
Pt CI
H3N
CI
HO
Cisplatin
O
O
H3N
H3N
O
O Carboplatin
DCA
CI CI
DCA-Pt(II)
pH=7.4, [CI−]= 100 mmol/l
(b)
pH=5.0, [CI−]= 4−10 mmol/l
II)
t( A-P
DC
ed
n
atio
Aqu
DCA releas
Mitochondrion
CI H 2O Pt
H3N
O
A
d
se
ea
rel
DC
Depolarization
tion
Aqua
H 3N
CI
O
NH3 H2O H3N
Pt
Pt NH3
H2O DNA binding
H3N
g
kin
slin
ros
C
Cancer cells Possible mechanism of action of DCA-Pt(II) in cancer cells. (a) Cisplatin, DCA, carboplatin, and DCA-Pt(II); (b) DCA-Pt(II), passively diffusing into the cancer cells, undergoes aquation, facilitating the release of two DCA molecules and aquated cisplatin. Thereafter, DCA depolarizes the mitochondrion, whereas cisplatin crosslinks with DNA. The concerted action reverses cisplatin resistance. DCA, dichloroacetate; Pt, platinum.
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700 Anti-Cancer Drugs 2015, Vol 26 No 7
and dried under vacuum. A weight of 0.42 g of the product was obtained in an 87% yield. Cell lines and culture conditions
A2780 and A2780DDP cells were purchased from the Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, Shanghai, China, and grown in RPMI-1640 medium (Life Technologies) supplemented with 10% fetal bovine serum, 0.03% L-glutamine, and 1% penicillin/streptomycin at 37°C in a 5% CO2 atmosphere. LO2 human liver cells were purchased from the American Type Culture Collection and cultured in RPMI-1640 medium supplemented with 10% newborn calf serum, 0.03% L-glutamine, and 1% penicillin/streptomycin at 37°C in a 5% CO2 atmosphere. MTT assay
A2780 and A2780DDP cells harvested in the logarithmic growth phase were seeded in 96-well plates at a density of 1 × 104 cells/well and incubated with RPMI-1640 medium for 12 h. The medium was then replaced with RPMI-1640 medium containing either (i) cisplatin, cisplatin + 2DCA, and DCA-Pt(II) at a final equivalent Pt concentration of 0.2–108 μmol/l, (ii) carboplatin and carboplatin + 2DCA at a final equivalent Pt concentration of 0.2–432 μmol/l, or (iii) DCA at a two concentration ranges (0–200 μmol/l and 312.5–50 mmol/l), which was used as a control. The incubation time for all drugs was 48 h. After incubation, 20 μl of MTT solution in PBS with a concentration of 5 mg/ml was added to the wells, and the plates were incubated for another 4 h at 37°C, followed by removal of the culture medium containing MTT and addition of 150 μl dimethyl sulfoxide to each well to dissolve the formazan crystals formed. Finally, the plates were shaken for 10 min, and the absorbance of the formazan product was measured at 492 nm on a microplate reader. Determination of Pt content in the cancer cells
A2780 and A2780DDP were seeded in six-well plates at a density of 1 × 106 cells/well. At their logarithmic phase of growth, the cells were treated with cisplatin, cisplatin + 2DCA, DCA-Pt(II), carboplatin, and carboplatin + 2DCA, with final Pt concentrations in the culture medium regulated to 10 μmol/l, and incubated at 37°C for 1 and 4 h, respectively. As described previously [14,16,17], to remove surface-bound drugs the cells were washed three times with ice-cold PBS, incubated with 1.5 ml of 0.15 mol/l sodium chloride (pH 3.0 was adjusted by acetic acid) for 3 min at 4°C, rinsed with 2 ml of cold PBS, harvested by scraping in ice-cold PBS, and centrifuged. Thereafter, the cell pellets were lysed by adding 200 μl of cell lysis buffer (Promega, Beijing, China), and the cell lysis solution was frozen at − 20°C for 20 min and thawed at room temperature. For each sample, 100 μl of the cell lysis solution was used to directly measure the Pt content by inductively coupled plasma mass spectrometry (ICPMS). The other 100 μl of the cell lysis solution was used
to determine the protein content in each cell sample using the bicinchoninic acid protein assay kit (Beyotime Institute of Biotechnology, Shanghai, China) according to previously published data [14,16,17]. The Pt content was expressed as nanograms of Pt per milligram of total proteins. Determination of Pt-DNA adduct contents in the cancer cells
A2780 and A2780DDP were seeded in six-well plates at a density of 1 × 106 cells/well. At their logarithmic phase of growth, the cells were treated with cisplatin, cisplatin + 2DCA, DCA-Pt(II), carboplatin, and carboplatin + 2DCA, with the final Pt concentration in the culture medium regulated to 2 μmol/l, and incubated at 37°C for 24 h. Next, we used a genomic DNA extraction kit (Tiangen Biotech, Beijing, China) to isolate genomic DNA from the cells treated with the different drugs, and we finally added 200 μl distilled water to dissolve the DNA. Thereafter, we used Nano drop to measure the concentration of DNA with 1 μl DNA. The remaining 200 μl of DNA was diluted to 1 ml in volume. The Pt content was measured by ICP-MS and expressed as nanograms of Pt per milligram of total genomic DNA. MMP analysis
It is well known that MMP (Δψm) is an important parameter in the mitochondrial function of cells. JC-1 (Beyotime Biotech; 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethyl-imidacarbocyanine iodide) is a cationic and lipophilic dye that can enter the mitochondria and change its fluorescence color on the basis of a change in the MMP (Δψm), as it exists in the monomeric form at low Δψm, at which it shows green fluorescence (∼520 nm), and it exists as ‘Jaggregates’ at high Δψm, which fluoresce red (∼590 nm). Therefore, it is often used to characterize MMP. After the cells are stained with JC-1, their green fluorescence and red fluorescence are detected, respectively, and the intensity ratio of green/red fluorescence is considered as a measure of Δψm of the cells. On the basis of the green/red ratio, mitochondrial function or cellular function can be deduced. In the present study, A2780 and A2780DDP cells were seeded into black 96-well microplates. After 24 h, the drugs [cisplatin, cisplatin + 2DCA, DCA-Pt(II), carboplatin, and carboplatin + 2DCA at 50 μmol/l Pt and 100 μmol/l DCA concentrations] were added to the wells for 1 h incubation, and the cells without treatment were used as controls. At the end of drug treatment, the medium was removed and the cells were incubated at 37°C for 1 h with 5 mg/l of JC-1. Thereafter, they were washed twice with PBS and placed in fresh medium without serum. Following this, images were viewed by high-throughput fluorescence confocal microscopy (BD Pathway Bioimager 855; BD Biosciences, San Diego, California, USA) at an excitation wavelength of 488 nm and an emission wavelength of 530 nm for green, and at an excitation wavelength of 540 nm and an emission wavelength of 590 nm for red fluorescence. The intensities
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DCA-Pt(II) overcomes cisplatin drug resistance Zhang et al. 701
Fig. 1
(a)
(b)
100
500 A2780
A2780
431.85
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404.75
400
A2780DDP
IC50 values (μmol/l)
60
40
20
338.5 317.4
300 200 84.5
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18.9
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pl at
ar
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in
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Effects of various drugs on cisplatin-sensitive ovarian cancer cells, A2780, and cisplatin-resistant cells, A2780DDP (mean ± SD, n = 3). (a) Representative dose-dependent cell viability curve of cisplatin and (b) IC50 of various drugs. Data above the columns are the IC50 values based on Pt read. For the tested drugs cisplatin + 2DCA and carboplatin + 2DCA, the molar ratio of DCA to Pt is 2 : 1. DCA alone at a concentration less than 10 mmol/l shows no difference in the inhibition of A2780 and A2780DDP cancer cell lines (IC50 of DCA is 33 and > 50 mmol/l in A2780 and A2780DDP, respectively; Fig. S2, Supplemental digital content 1, http://links.lww.com/ACD/A104). DCA, dichloroacetate; IC, inhibitory concentration; Pt, platinum.
of both green fluorescence (JC-1 monomers) and red fluorescence (J-aggregates) were measured using a FLUOstar optima plate reader (BMG Labtech, Offenburg, Germany), with the filters set to 485 nm excitation/520 nm emission (green) and 544 nm excitation/590 nm emission (red), and the data were presented as the ratio of green to red signals (520/590 nm), which was normalized to that for the nontreated cells.
Western blot of cleaved-caspase-3 and cleaved-caspase-9
A2780 and A2780DDP were seeded in six-well plates at a density of 1 × 106 cells/well. At their logarithmic phase of growth, the cells were treated with drugs [cisplatin, cisplatin + 2DCA, DCA-Pt(II), carboplatin, and carboplatin + 2DCA at 50 μmol/l Pt and 100 μmol/l DCA concentrations] and incubated at 37°C for 10 h. Thereafter, the cells were harvested and rinsed twice with PBS. Cell extracts were treated with lysis buffer [1% Nonidet P-40, 50 mmol/l Tris-HCl (pH 7.5), 150 mmol/l NaCl, 1 mmol/l NaF, 1 mmol/l phenylmethylsulfonyl fluoride, 4 μg/ml leupeptin, and 1 μg/ml aprotinin] for 30 min with occasional rocking, followed by centrifugation at 15 000 rpm for 15 min at 4°C. Identical amounts (100 μg of protein) of cell lysate were resolved by 10% SDS-PAGE, transferred onto a PVDF membrane, and then blocked with 5% fat-free dry milk in TBST [20 mmol/l Tris-HCl (pH 7.6), 150 mmol/l NaCl, and 0.02% Tween-20] for 1 h at room temperature. The
membrane was immunoblotted with rabbit anti-human cleaved-caspase-3 and cleaved-caspase-9 polyclonal antibodies (1 : 1000; Cell Signaling, Boston, Massachussetts, USA) in 1% milk/TBST. To assure equivalent protein loading, the membranes were simultaneously incubated with GAPDH monoclonal antibodies (1 : 1000; Kangcheng Co., Shanghai, China) at 4°C overnight. The membranes were then probed with diluted primary antibodies in 1% milk/TBST for 24 h at 4°C, washed three times, incubated with horseradish peroxidase-conjugated secondary antibodies for 30 min at room temperature, and washed extensively before detection by chemiluminescence using the ECL-Plus kit (Beyotime Biotech). Proteins were visualized by exposing the blots to Kodak film (Eastman Kodak, Rochester, New York, USA). Western blotting data were quantified using Image J software (National Institutes of Health, Bethesda, Maryland, USA).
Cell cycle analysis
Flow cytometry was used to evaluate the number of cells in specific phases of the cell cycle. Cell cycle distribution was determined by staining DNA with propidium iodide. A2780 and A2780DDP were seeded in six-well plates at a density of 1 × 106 cells/well. At their logarithmic phase of growth, drugs [cisplatin, cisplatin + 2DCA, and DCA-Pt (II) at 20 μmol/l Pt concentration] were added to the wells and the cells were incubated for 16 h; cells without treatment were used as controls. After treatment, cells were collected, washed twice with ice-cold PBS buffer
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702 Anti-Cancer Drugs 2015, Vol 26 No 7
Fig. 2
Intracelluar uptake (ng Pt/mg protein)
(a)
300 250
1 h A2780
1 h A2780DDP
4 h A2780
4 h A2780DDP
200 150 100 50
C
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in
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Intracellular uptake and Pt-DNA adducts (mean ± SD, n = 3). (a) Drug uptake by A2780 and A2780DDP cells over 1 and 4 h. (b) Pt-DNA adducts of drugs in A2780 and A2780DDP cells incubated for 24 h. DCA, dichloroacetate; Pt, platinum.
(pH 7.4), fixed with 70% alcohol at 4°C overnight, and then stained with propidium iodide (1 mg/ml) in the presence of 1% RNAase A at 37°C for 30 min. The percentages of cells in different phases of the cell cycle were measured using a flow cytometer (Beckman Coulter Epics; Beckman Coulter, Pasadena, California, USA), and the percentages of cells in G0/G1, S, and G2/M phases were analyzed using MultiCycle software (Microsemi Corp., Aliso Viejo, California, USA). Statistical analysis
The data were expressed as mean ± SD. Student’s t-test was used to determine the statistical difference between various experimental and control groups. Differences were considered statistically significant at P less than 0.05.
Results and discussion Cell viability studies
We show a representative dose-dependent cell viability curve of cisplatin for the epithelial ovarian cancer cell line A2780 and its acquired resistant cell line A2780DDP (Fig. 1a). A2780DDP shows a much more inert response to cisplatin, and the cell viability was more than 20% even at the highest concentration of 200 µmol/l, indicating the inability of cisplatin to kill the cancer cells, which
may be the major cause of clinical tumor recurrence. The IC50 of cisplatin for A2780 and A2780DDP were 22.1 and 84.5 µmol/l, respectively, indicating a resistance of ∼ 3.8-fold (Fig. 1b). DCA does not markedly inhibit the two cell lines even at a concentration of up to 10 mmol/l (Fig. S2, Supplemental digital content 1, http:// links.lww.com/ACD/A104). To test the efficacy of DCA-Pt (II) in reversing drug resistance, a mixture of cisplatin and two molar equivalent DCA (cisplatin + 2DCA) and a mixture of carboplatin and carboplatin with two molar equivalent DCA (carboplatin + 2DCA) were chosen as controls. DCA-Pt(II) is found to be the most effective drug among all the tested groups in the two cancer cell lines. Its IC50 values were 18.9 and 21.35 µmol/l for A2780 and A2780DDP, respectively, implying a resistance of ∼ 1.1-fold, which is markedly lower than that of cisplatin (∼3.8-fold). Therefore, DCA-Pt(II) reversed cisplatin drug resistance by 3.5-fold. DCA-Pt(II) has more similarity to carboplatin in chemical structure as both share moderately liable leaving groups, that is, carboxylic acids, whereas cisplatin has two chloride ions as readily liable leaving groups. Therefore, to some extent, it makes more sense to compare DCA-Pt(II) with carboplatin and carboplatin + 2DCA. We found that DCA-Pt(II) is ∼ 18 and 19 times more effective than carboplatin, and 16.8 and 20.2 times more effective than carboplatin + 2DCA in A2780 and A2780DDP, respectively. The results indicate that DCA-Pt(II) is the most effective drug in both ovarian cancer cell lines and can help overcome drug resistance in ovarian cancer. An ideal anticancer drug should be less toxic to healthy cells. Therefore, we analyzed the toxicity of these drugs in normal liver cells (L02) over 6–24 h, which showed that DCA-Pt(II) has a lower toxicity than cisplatin and cisplatin + 2DCA under all conditions (Fig. S1, Supplemental digital content 1, http://links.lww.com/ACD/A104). Pt and Pt-DNA adduct content test in cells
Pt(II)-based drugs are believed to passively diffuse into cancer cells [18] and bind to cellular biomolecules (amino acid, proteins, RNA, DNA, etc.) after their subsequent intracellular dissociation. For cisplatin and carboplatin, it is generally believed that the efficacy of Pt(II) drugs is great determined by the amount of intracellular drug and the ultimate Pt-DNA adducts [14]. However, compared with A2780 cells, the difference in biological characteristics (lower levels of membrane protein copper transporter 1, higher levels of intracellular glutathione and DNA repair genes and proteins) of resistant A2780DDP cells makes them internalize fewer Pt drugs, and hence, fewer Pt-DNA adducts are formed [4–6]. To provide insight into the higher efficacy and possible reason for overcoming drug resistance of DCA-Pt(II), we first determined the intracelluar uptake of various drugs [cisplatin, DCA-Pt(II), cisplatin + 2DCA, carboplatin, and carboplatin + 2DCA] by A2780 and A2780DDP
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DCA-Pt(II) overcomes cisplatin drug resistance Zhang et al. 703
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DCA-Pt(II) induced mitochondria-mediated apoptosis in A2780 and A2780DDP cells. (a) The mitochondrial potential changes as revealed by the JC-1 assay; (b) A2780 cell lines; (c) A2780/DDP cell lines; (d) quantitative analysis of cleaved-caspase-3; (e) cleaved-caspase-9 of the two cell lines treated with various drugs. DCA, dichloroacetate; Pt, platinum; JC-1, 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethyl-imidacarbocyanine iodide.
during 1 and 4 h of drug incubation using ICP-MS. The results presented in Fig. 2a indicate that (i) the uptake of cisplatin by both A2780 and A2780DDP is time dependent. From 1 to 4 h, more drugs were taken up. Drug accumulation by drug-resistant A2780DDP cells is 48% of that by A2780 cells at 1 h (9.1 vs. 18.9 ng Pt/mg protein) and 42% at 4 h (99.4 vs. 239.1 ng Pt/mg protein); (ii) adding DCA to cisplatin/carboplatin to make a physical mixture seems to reduce the drug uptake (cisplatin vs. cisplatin + 2DCA; carboplatin vs. carboplatin + 2DCA), whereas fusion of DCA and cisplatin to one molecule of DCA-Pt(II) was also shown to reduce the drug uptake by both cell lines at 1 and 4 h. (iii) At 1 and 4 h, DCA-Pt(II) leads to the accumulation of the least amount of drugs in both cell lines, which is seemingly contradictory to the
fact that DCA-Pt(II) is the most effective and overcomes cisplatin resistance. Next, we tested the Pt-DNA adducts in the two cell lines after 24 h of drug incubation at an equal Pt concentration of 2 µmol/l (Fig. 2b). Consistent results can be found, as follows: (i) fewer Pt-DNA adducts were found in A2780DDP compared with A2780 cells for all the drugs; (ii) cells treated with cisplatin or cisplatin + 2DCA form many more Pt-DNA adducts than those treated with DCA-Pt(II), carboplatin, or carboplatin + 2DCA. This is due to the higher inertness of the carboxylic leaving group of carboplatin and DCA-Pt (II), whereas cisplatin has more liable chloride leaving groups; (iii) apart from drug groups with carboplatin, DCA-Pt(II) forms the fewest Pt-DNA adducts. Considering the fact that DCA-Pt(II) can overcome
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704 Anti-Cancer Drugs 2015, Vol 26 No 7
Table 1
Proportion of A2780 and A2780DDP cells in each phase of the cell cycle G0/G1 phase
Control Cisplatin Cisplatin + 2DCA DCA-Pt(II)
S phase
G2/M phase
A2780 (%)
A2780DDP (%)
A2780 (%)
A2780DDP (%)
A2780 (%)
A2780DDP (%)
66.85 ± 0.85 30.90 ± 0.65 31.49 ± 1.02 42.92 ± 1.33
55.48 ± 0.54 49.90 ± 0.90 50.60 ± 1.12 49.17 ± 1.36
22.11 ± 0.47 60.96 ± 0.32 63.26 ± 2.15 57.08 ± 1.89
28.66 ± 0.38 36.83 ± 0.85 36.87 ± 1.23 46.58 ± 1.40
11.05 ± 0.23 8.14 ± 0.58 5.24 ± 0.13 0.00 ± 0.12
15.86 ± 0.23 13.27 ± 0.47 12.53 ± 0.78 4.25 ± 0.12
Results represent the mean ± SD of three independent experiments. DCA, dichloroacetate; Pt, platinum.
cisplatin resistance with highest efficacy, the lower amount of Pt intracellular uptake and Pt-DNA adducts indicate other possible cellular pathways, unlike those involved with cisplatin, to kill more cancer cells.
Mitochondrion-dependent apoptosis research
DCA-Pt(II) has two DCA molecules; hence, it may exert some effect on the mitochondria. To find the possible mechanism for overcoming drug resistance, the cell mitochondrial transmembrane potential was measured using the established JC-1 assay [15]. JC-1 can selectively enter the mitochondria and reversibly change color from red to green as the membrane potential reduces. In healthy cells with high mitochondrial transmembrane potential, JC-1 forms complexes known as J-aggregates, which fluoresce intense red. In apoptotic cells with low mitochondrial transmembrane potential, JC-1 remains in the monomeric form, which fluoresces green. Therefore, a greater green/red ratio in fluorescence indicates more cell death. Figure 3a shows the green to red signals of cells treated with various drugs. DCA has an effect on the change in the red to green fluorescence ratio, indicating a high ability for reducing the mitochondrial membrane potential and inducing apoptosis. However, cisplatin and carboplatin lead to little alteration in the green to red ratio, suggesting limited effects on depolarization of the mitochondrion and inducing its dysfunction. Adding DCA to cisplatin (cisplatin + 2DCA) or carboplatin (carboplatin + 2DCA) resulted in a great change in the green/red fluorescence ratio, whereas DCA-Pt(II) seems to have the largest effect on depolarization of the mitochondrion and its dysfunction in both cell lines. We also found that DCA-Pt(II) had an even larger influence on the resistance A2780DDP compared with A2780. The results here indicate that DCA-Pt(II) has a great effect on the mitochondria, which mediates the cellular apoptotic pathway. This differs from the present Pt drugs and is possibly the reason that DCA-Pt(II) is much more effective than other drugs in the two cell lines and that DCA overcomes cisplatin drug resistance. In mitochondrion-dependent apoptosis, exposure of cells to various stress signals triggers the disruption of the mitochondrion, leading to the release of cytochrome c into the cytosol [19]. The apoptosomes formed contain cytochrome c, Apaf-1, and caspase-9. The activation of
caspase-9 leads to the cleavage and activation of executioner caspase-3, leading to apoptosis [20]. Hence, we test the activation of caspase-9 and caspase-3 in A2780 and A2780DDP cells treated with different drugs. As shown in Fig. 3b and c, cisplatin can induce activation of caspase-3 to a larger extent in A2780 cells than in A2780DDP cells. In the presence of DCA, the effects of cisplatin in A2780DDP cells seems better. However, DCA-Pt(II) had the largest effect on caspase-3 in both A2780 parental cells and A2780DDP resistant cells. We can also see a clear difference in cleaved-caspase-3 levels between A2780 and A2780DDP cells in quantitative analysis (Fig. 3d), further suggesting drug resistance of A2780DDP cells to cisplatin. As caspase-9 acts upstream of caspase-3 and activates caspase-3 leading to apoptosis, we measure the activation of caspase-9 induced by Pt drugs. We found that adding DCA to cisplatin (cisplatin + 2DCA) and carboplatin (carboplatin + 2DCA) both resulted in the activation of caspase-9 in A2780DDP cells, whereas DCA-Pt(II) induced more obvious activation of caspase-9 in both A2780 cells and A2780DDP cells (Fig. 3e). We also found that DCA can induce more activation of caspase-9, which makes A2780DDP cells more sensitive to cisplatin, but has no effect on the activation of caspase-3. Therefore, these data above suggest that DCA-Pt(II) is much more effective than other drugs especially in A2780DDP cells, which is possibly related to caspase-9-mediated mitochondrial dysfunction, which further leads to cell apoptosis.
Effect of platinum drugs on cell cycle distribution
The cell cycle is typically divided into different phases: quiescent and gap 1 (G0/G1 phase), synthesis (S phase), and gap 2 and mitosis (G2/M phase), which governs the transition from quiescence through cell growth to proliferation. To determine whether the effects of Pt drugs on proliferation are mediated by inhibition of cell cycle progression, the cell cycle phase distribution was analyzed in A2780 and A2780DDP cells by flow cytometry. Cells were treated with drugs [cisplatin, cisplatin + 2DCA, DCA-Pt(II) at a Pt concentration of 20 μmol/l] for 16 h. As shown in Table 1, incubation of A2780 cells leads to redistribution of the percentage of cells in the phases of the cell cycle. Cisplatin alone arrested A2780 and
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DCA-Pt(II) overcomes cisplatin drug resistance Zhang et al. 705
A2780DDP cells at the S phase. When the cells were treated with ‘cisplatin + 2DCA’, the percentage of both cell lines in the G2/M phase decreased by 6 and 3%, respectively. The results indicate that DCA can promote cell cycle arrest induced by cisplatin. In particular, no A2780 cells stayed in the G2/M phase after DCA-Pt(II) treatment, and only 4.25% of A2780DDP cells stayed in the G2/M phase with the same treatment. A 35% increase in A2780 cells in the S phase and a 11% decrease in A2780 cells in the G2/M phase were seen on treatment with DCA-Pt(II). At the same time, there was an 18% increase in A2780DDP cells in the S phase and an ∼ 11% decrease in A2780DDP cells in the G2/M phase after treatment with DCA-Pt(II). These results suggest that the DCA-Pt(II) blocked cell cycle progression more effectively than other drugs. Conclusion
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Here, we found that DCA-Pt(II), which is different from clinical Pt drugs used currently, overcomes ovarian cancer drug resistance by dual organelle targeting. Despite less cellular uptake and fewer Pt-DNA adducts present, DCAPt(II) induces much more mitochondrial dysfunction, as well as caspase-3 and caspase-9 cleavage, thereby resulting in greater levels of apoptosis in both sensitive and resistant cell lines. Furthermore, DCA-Pt(II) induced S phase arrest in A2780 parental cells and A2780DDP resistant cells. Although the detailed mechanism of action requires further investigation, the concerted manner of targeting different organelles makes DCA-Pt(II) a candidate for further study, especially in drug-resistant cancer.
Acknowledgements This work is supported by the National Natural Science Foundation of China (Nos. 51403031, 31170324, and 31070318), the China Postdoctoral Science Foundation (No. 2014M550163), the Research Funds from Science & Technology Department of Jilin Province (Nos. 20140520049JH, and 20130201008ZY), and the funds from the Science & Technology Department of Changchun City (No. 2012098).
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Conflicts of interest
There are no conflicts of interest.
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