Conceived and designed the experiments: BS SR MG. Performed the experiments: BS MH VM SR JPG. Analyzed the data: BS MH VM SR JPG MG. Contributed reagents/materials/analysis tools: JJO MC. Wrote the paper: BS.
The authors have declared that no competing interests exist.
The IGROVCDDP cisplatin-resistant ovarian cancer cell line is also resistant to paclitaxel and models the resistance phenotype of relapsed ovarian cancer patients after first-line platinum/taxane chemotherapy. A TaqMan low-density array (TLDA) was used to characterise the expression of 380 genes associated with chemotherapy resistance in IGROVCDDP cells. Paclitaxel resistance in IGROVCDDP is mediated by gene and protein overexpression of P-glycoprotein and the protein is functionally active. Cisplatin resistance was not reversed by elacridar, confirming that cisplatin is not a P-glycoprotein substrate. Cisplatin resistance in IGROVCDDP is multifactorial and is mediated in part by the glutathione pathway and decreased accumulation of drug. Total cellular glutathione was not increased. However, the enzyme activity of GSR and GGT1 were up-regulated. The cellular localisation of copper transporter CTR1 changed from membrane associated in IGROV-1 to cytoplasmic in IGROVCDDP. This may mediate the previously reported accumulation defect. There was decreased expression of the sodium potassium pump (ATP1A), MRP1 and FBP which all have been previously associated with platinum accumulation defects in platinum-resistant cell lines. Cellular localisation of MRP1 was also altered in IGROVCDDP shifting basolaterally, compared to IGROV-1. BRCA1 was also up-regulated at the gene and protein level. The overexpression of P-glycoprotein in a resistant model developed with cisplatin is unusual. This demonstrates that P-glycoprotein can be up-regulated as a generalised stress response rather than as a specific response to a substrate. Mechanisms characterised in IGROVCDDP cells may be applicable to relapsed ovarian cancer patients treated with frontline platinum/taxane chemotherapy.
The prognosis for women with ovarian cancer is very poor. The majority of patients present with advanced disease and the long-term survival in these patients is 10–30%
The IGROVCDDP cisplatin-resistant ovarian cell line is an unusual cisplatin-resistant model, as it is also cross-resistant to paclitaxel. When acquired cisplatin resistance is produced in cell lines, only 17% are also resistant to paclitaxel
IGROVCDDP models the resistance phenotype of ovarian cancer patients who have failed standard frontline combination platinum/taxane chemotherapy. Chemotherapeutic drugs which IGROVCDDP is sensitive to may be suitable for the treatment of platinum/taxane resistant ovarian cancer. Studying the IGROVCDDP drug-resistant model will allow us to understand the mechanisms of cross resistance between platinums and taxanes. It is our aim to translate molecular markers of this cross resistance phenotypes to the clinical treatment of relapsed drug-resistant ovarian carcinoma.
The human IGROV-1 ovarian cancer cell line and its cisplatin-resistant variant IGROVCDDP were obtained from Prof. Jan Schellens
Cells (1.25×106 cells/10 cm dish) were plated and allowed to attach and grow for 3 days to reach 70–80% confluence. The cells were then trypsinised, washed in 10 mL PBS, centrifuged and the supernatant removed. The cell pellets were stored at −80°C prior to analysis. Total RNA was prepared using a RNeasy Mini Kit (Qiagen, UK). The TLDA array was performed on biological triplicate samples as described in Gillet
Cells were plated at a density of 2.5×105 in a non-vented T25 flask. The next day the media was removed and the cells were treated with 1 µM epirubicin for 2 hours in the presence or absence of 0.25 µM elacridar, 0.67 µM, 3.33 µM or 33.3 µM cisplatin. Cells were washed with 4 mL of cold PBS and trypsinised. The cells were centrifuged and resuspended in 1 mL of PBS and a cell count performed (9 µL). The remaining cells were centrifuged, supernatant removed and the pellet stored at −20°C prior to analysis. Total epirubicin was then quantified by LC-MS following a liquid-liquid extraction sample preparation, according to the method of Wall
Cells were plated at a density of 1.25×106 cells in a 10 cm diameter dish and allowed to attach overnight. The cells were drug treated for 24 hours, then trypsinised and a cell count performed. The cells were washed in 10 mL PBS, centrifuged and the supernatant removed. The cell pellets were stored at −20°C prior to analysis. Total glutathione was determined using a modification of Suzakake
Cell culture - Cells (6.25×105 cells/10 cm dish) were plated and allowed to attach overnight. Cells were then treated with 0.67 µM cisplatin. Drug-treated cells and their controls were trypsinised and a cell count performed. The cells were then washed in 10 mL PBS, centrifuged and the supernatant removed. The pellet was resuspended in 400 µL cold enzyme assay buffer (100 mM potassium phosphate monobasic, 100 mM EDTA; pH 7.5). 16 µL of 25× Complete Protease Inhibitor (Roche, UK) was added, and the sample was sonicated. After centrifugation (13000 rpm for 15 minutes at 4°C) the supernatant was collected and frozen at −80°C prior to analysis.
GSR – GSR (Sigma) standards were made up in glutathione reductase dilution buffer (100 mM potassium phosphate monobasic; 100 mM EDTA; 1 mg/mL BSA; pH 7.5) ranging from 0.3–0.0037 units/mL. 40 µL of each sample and standard were assayed in duplicate in 96 well plates. The reaction mix was then added 160 µL total volume (2 mM oxidised glutathione (100 µL); 3 mM DNTB (50 µL); 2 mM NADPH (10 µL)).
GGT1
Analysis - The plates were read at 412 nM (preheating to 30°C) and 405 nM (preheating to 37°C) for GSR and GGT1 respectively, with kinetic measurement by a plate reader as described for the glutathione assay.
Cells (1.25×106 cells/10 cm dish) were plated and allowed to attach overnight. The cells were then drug-treated with cisplatin and grown for 3 days. Cells were resuspended in 100 µL lysis buffer (0.01 M Tris/HCl, pH 7.4) and sonicated. 20 µg of protein was diluted in Laemmli sample buffer, boiled for 3 minutes, cooled on ice and loaded onto 12% Tris/glycine gels with a 4% stacking gel. Samples and molecular weight markers were then electrophoresised for 90 minutes at 100 V. The gels were electrotransferred to 0.45 µm nitrocellulose membranes (Biorad) for 90 minutes at 100 V using a wet transfer system (Biorad). The membranes were blocked with 5% non-fat skim milk (Biorad) in PBS for 2 hours, then incubated with the primary antibody prepared in 3% skim milk/0.1% tween/PBS (
Protein | kDa | Host | Supplier | Catalogue # | Dilution Western | Dilution Confocal |
ATP1A1 | 110 | Mouse | Abcam | ab2872 | 1∶250 | N/A |
ATP7A | 180 | Rabbit | Gift from Prof. Anthony Monaco as described |
1∶1000 | N/A | |
BCRP | 72 | Mouse | Alexis | ALX-801-029-C250 | 1∶250 | N/A |
BRCA1 | 220 | Rabbit | Cell Signalling Technology | 9010 | 1∶500 | N/A |
β-Actin | 42 | Mouse | Sigma | A5441 | 1∶10,000 | N/A |
CTR1/SLC31A1 | 30 | Rabbit | Novus | NB100-402 | 1∶1000 | 1∶250 |
FBP | N/A | Rabbit | Novus | NBP1-32293 | N/A | 1∶250 |
GM130 | N/A | Mouse | Transduction Labs | 610823 | N/A | 1∶500 |
GCLC (γGCS) | 73 | Mouse | Abcam | ab55435 | 1∶500 | N/A |
GGT1 | 61.4 | Mouse | Sigma | WH0002678M1-100UG | 1∶1000 | N/A |
GSR | 56.2 | Mouse | Sigma | WH0002936M1-100UG | 1∶1000 | N/A |
MRP1 | 190 | Rat | Alexis | ALX-801-007-C250 | 1∶250 | 1∶250 |
MRP2 | 180 | Mouse | Alexis | ALX-801-016-C250 | 1∶250 | N/A |
P-glycoprotein | 170 | Mouse | Alexis | ALX-801-002-C100 | 1∶250 | N/A |
Anti-Mouse HRP | N/A | Sheep | Sigma | A6782 | 1∶1000 | N/A |
Anti-Rabbit HRP | N/A | Goat | Sigma | A4914 | 1∶1000 | N/A |
Anti-Mouse AP | N/A | Rabbit | Sigma | A4312 | 1∶1000 | N/A |
Anit-Rat Alexa488 | N/A | Donkey | Invitrogen | A21208 | N/A | 1∶500 |
Anti-Rabbit Alexa488 | N/A | Goat | Invitrogen | A11008 | N/A | 1∶500 |
Anti-Mouse Alexa594 | N/A | Goat | Invitrogen | A11005 | N/A | 1∶500 |
AP – Alkaline Phosphatase, ATP1A1 - Na+/K+ transporting alpha 1, ATP7A - ATPase, Cu++ transporting, alpha polypeptide, BCRP - Breast Cancer Resistance Protein, BRCA1 - Breast Cancer Susceptibility Protein 1, CTR1 - solute carrier family 31 (copper transporters), member 1, FBP – Folate Binding Protein, GM130 - Golgin A2, γGCS – gamma Glutamyl Cysteine Synthesase, GSR - Glutathione Reductase, GGT1 - Gamma Glutamyl Transpeptidase, HRP – Horseradish Peroxidase, MRP1 - Multidrug resistance-associated protein-1, MRP2 - Multidrug resistance-associated protein-2.
Cells (1.5×105 cells/well) were plated into 8-well chamber slides and allowed to attach overnight. All washes were with PBS and all incubations were at room temperature unless otherwise specified. The cells were washed twice, and fixed with 4% paraformaldehyde (Sigma) in PBS for 30 minutes at 37°C. The cells were permabilised with 0.5% Triton-X-100 (Sigma) for 10 minutes and washed twice. Cells were stained with a 50 µg/mL fluorescent TRITC solution in PBS for 40 minutes and then washed twice. The cells were then incubated with blocking buffer (0.02% BSA in PBS) for 30 minutes at 37°C. The cells were then incubated with primary antibody (
All experiments were performed at minimum in triplicate. Two-sample, two tailed student’s t-tests were used to determine significant differences using p<0.05 as a cut off.
The IGROV-1 and IGROVCDDP cells were analysed for 380 genes associated with chemoresistance by TLDA array in order to characterise the mechanisms of platinum and taxane resistance. 145 genes were found to be significantly different between IGROV-1 and IGROVCDDP based on a p<0.01 cutoff. Genes chosen for further analyses were based on the most significant by p-value as well as those pathways previously associated with platinum and taxane resistance (
The gene expression of P-glycoprotein (P-gp) is increased in IGROVCDDP (
A) Western blot of P-glycoprotein, IGROV-1 (open bars) and IGROVCDDP (grey bars) with and without treatment with 0.67 µM cisplatin for 72 hours (striped bars). Representative image shown. Graph shows quantitation of n = 6 biological repeats normalised to β-actin. * Indicates significant difference from IGROV-1 p<0.05 student’s t-test. B) Accumulation of epirubicin determined by LC-MS. IGROV-1 (open bars) and IGROVCDDP (shaded bars). Cells were treated with 1 µM epirubicin for 2 hours, 0.25 µM elacridar, 0.67 µM, 3.33 µM or 33.3 µM cisplatin were investigated as modulators of epirubicin accumulation. Graph shows quantitation of n = 3 biological repeats normalised to cell number. * Indicates a significant difference between IGROV-1 and IGROV-CDDP, # Indicates a significant difference on the addition of a modulator (p<0.05 students t-test). C) Cytotoxicity of IGROV-1 and IGROVCDDP to P-glycoprotein and non P-glycoprotein substrates.
Gene | Full Name/Synonyms | Mean mRNA Fold Change | SD | P-value | ||
|
||||||
ABCB1 | P-glycoprotein |
|
11.38 | 0.45 | 2.29E–06 | |
ABCG2 | BCRP/Breast Cancer Resistance Protein |
|
−2.17 | 0.19 | 2.95E–02 | |
|
||||||
ABCC2 | MRP2 cMOAT |
|
−3.27 | 0.07 | 1.15E–03 | |
|
||||||
ATP1A1 | Na+/K+ transporting alpha 1 |
|
−4.52 | 0.02 | 1.09E–05 | |
ABCC1 | MRP1 |
|
−1.43 | 0.02 | 3.15E–04 | |
|
||||||
GSR | Glutathione Reductase |
|
1.40 | 0.12 | 1.24E–02 | |
GGT1 | Gamma Glutamyl Transpeptidase |
|
4.92 | 1.75 | 5.90E–04 | |
|
||||||
BRCA1 | Breast Cancer Susceptibility Protein 1 |
|
2.17 | 0.25 | 1.31E–03 |
IGROVCDDP cells were screened for their response to a variety of chemotherapeutics (
Drug (Units) | IGROV-1 IC50 | IGROVCDDP IC50 | Resistant vs Sensitive | IGROV-1+/− Ouabain | IGROVCDDP +/− Ouabain | ||
Mean ± SD | Mean ± SD | Fold | P-value | n | P-value | P-value | |
|
|||||||
Cisplatin (µM) | 0.14±0.06 | 2.70±1.15 | 18.73 |
|
15 | ||
+ Ouabain 0.01 nM | 0.07±0.00 | 1.47±0.72 | 21.69 |
|
3 | 0.226 | 0.073 |
|
|||||||
Ouabain (nM) | 23.84±9.11 | 3.38±0.59 | 0.14 |
|
3 |
The impact of co- or pre-treatment with cisplatin on paclitaxel cytotoxicity was investigated and no significant change was observed (data not shown). Similarly, co- or pre-treatment with paclitaxel did not reverse cisplatin resistance (data not shown).
Platinum resistance is associated with an intracellular shift of platinum uptake transporter CTR1 not resistance mediated by MRP2.
A) Cytotoxicity of IGROV-1 and IGROVCDDP to CuSO4. B) ATP7A western blot. Open bars are IGROV-1, shaded bars are IGROVCDDP and striped bars indicate treatment with 0.67 µM cisplatin for 72 hours. Representative image shown. Graph shows quantitation of n = 4 biological repeats normalised to β-actin. C) CTR1 western blot. Representative image shown. Graph shows quantitation of n = 3 biological repeats normalised to β-actin. * Indicates significant difference from IGROV-1 p<0.05 student’s t-test. CTR1 confocal microscopy in D) IGROV-1 and E) IGROVCDDP cells. XY planes are shown for DAPI (blue), Golgi (red) and CTR1 (green), a merged image is also shown.
MRP2, a transporter which can efflux cisplatin conjugates, had decreased gene expression in IGROVCDDP (
One of most significant differentially expressed genes in IGROVCDDP was a decrease in expression of the Na+/K+ pump (ATP1A1) (
Open bars are IGROV-1, shaded bars are IGROVCDDP and striped bars indicate treatment with 0.67 µM cisplatin for 72 hours. A) ATP1A1 western blot. Representative image shown. Graph shows quantitation of n = 4 biological repeats normalised to β-actin. B) MRP1 western blot. Representative image shown. Graph shows quantitation of n = 3 biological repeats normalised to β-actin. * Indicates significant difference from IGROV-1 p<0.05 student’s t-test. MRP1 confocal microscopy in C) IGROV-1 and D) IGROVCDDP cells. Orthogonal images are shown for a merged image of DAPI (blue) and MRP1 (green), arrows on the side bars indicate the apical (IGROV-1) and basolateral location of MRP1 (IGROVCDDP). FBP confocal microscopy in E) IGROV-1 and F) IGROVCDDP cells. XY planes are shown for DAPI (blue), actin (red) and FBP (green), a merged image is also shown.
Previous research has shown decreased expression and an intracellular shift of membrane proteins MRP1 and FBP to be associated with a defect in platinum accumulation in cisplatin-resistant cell lines
Open bars are IGROV-1, shaded bars are IGROVCDDP and striped bars indicate treatment with 0.67 µM cisplatin. A) Total intracellular glutathione. Graph shows n = 3 biological repeats normalised to cell number. B) γGCS western blot. Representative image shown. Graph shows quantitation of n = 4 biological repeats normalised to β-actin. C) GSR western blot. Representative image shown. Graph shows quantitation of n = 3 biological repeats normalised to β-actin. D) GSR enzyme assay. Graph shows n = 4 biological repeats normalised to cell number. E) GGT1 western blot. Representative image shown Graph shows quantitation of n = 3 biological repeats normalised to β-actin. E) GGT1 enzyme assay. Graph shows n = 4 biological repeats normalised to cell number. * Indicates significant difference from IGROV-1 p<0.05 student’s t-test. # Indicates significant difference from IGROVCDDP on the addition of cisplatin. G) Modulation of cisplatin cytotoxicity of IGROV-1 and IGROVCDDP with BSO.
The mRNA expression of glutathione reductase (GSR) and gamma glutamyl transpeptidase (GGT1) were both significantly increased in IGROVCDDP (
Open bars are IGROV-1, shaded bars are IGROVCDDP and striped bars indicate treatment with 0.67 µM cisplatin for 72 hours. A) BRCA1 western blot. Representative image shown. Graph shows quantitation of n = 4 biological repeats normalised to β-actin. * Indicates significant difference from IGROV-1 p<0.05 student’s t-test.
IGROVCDDP cells do not have higher levels of glutathione, and levels are not increased with low-level cisplatin treatment (
Parent Cell Line | Cancer | Cisplatin IC50 | ResistantCell Line | Cisplatin IC50 | Cisplatin FoldResistance | P-glycoprotein | Reference | |||
↑DNA | ↑mRNA | ↑Protein | ↑Activity | |||||||
LoVo | Colon | 1.30±0.11 µg/ml | CP2.0 | 8.30±0.12 µg/ml | 6.4 | No | No | Yes | Yes |
|
SKOV3 | Ovarian | 1.19±0.03 mM | SKOV3/CIS | 4.42±0.3 mM | 3.71 | ND | ND | Yes | Yes |
|
SKOV3 | Ovarian | 0.63±0.06 µM | SKOV3/DDP | 9.92±0.34 µM | 15.84 | ND | ND | Yes | ND |
|
SNU-601 | Gastric | 0.19 µg/ml | SNU-601/Cis2 | 9.2 µg/ml | 48.42 | ND | Yes | ND | Yes |
|
SNU-601/Cis10 | >100 µg/ml | >526 | ND | Yes | ND | No | ||||
WS | Lymphoma (Rat) | 900±30 nM | WR | 7100±1000 nM | 7.88 | ND | ND | Yes | Yes |
|
ND – Not Determined.
Increased expression of the DNA repair gene BRCA1 has been previously associated with cisplatin resistance
Resistance to paclitaxel in IGROVCDDP cells is mediated by an overexpression of P-gp at the gene (
Many models of acquired drug resistance will have overexpression of an transporter which effluxes the drug that was used to develop the model. Colchicine, a P-gp substrate
IGROVCDDP is the only cisplatin-resistant model developed from IGROV-1 known to overexpress P-gp and consequently have a platinum/taxane-resistant phenotype. Cisplatin-resistant models IGROV-1/Pt0.5 and IGROV-1/Pt1
Platinum resistance in the IGROVCDDP cells is multifactorial and involves the glutathione pathway and decreased accumulation of drug. This could result either from a complex regulatory pathway which controls many different mechanisms for conferring resistance to cisplatin, or could reflect the fact that the cells were selected in multiple steps and could therefore have accumulated different mechanisms at each step.
IGROVCDDP cells are low-level resistant to CuSO4 suggesting a role of copper transport in platinum resistance (
Our results show that while total cellular glutathione is not increased in IGROVCDDP (
Platinum accumulation defects mediated by decreased expression of ATP1A1 have been shown in H4-II-E/CDDP cisplatin resistant rat hepatoma cells
A mechanism has previously been described in cisplatin-resistant cell lines with platinum accumulation defects (KB-CP20 and 7404-CP20) in which surface expression of transporters is reduced, and some are overexpressed within cytoplasmic vesicles
The IGROVCDDP cells have increased mRNA (
IGROV-1/Pt0.5 and IGROV-1/Pt1, platinum-resistant and taxane-sensitive cells, have increased cellular GSH and decreased GGT1 enzyme activity
Decreased expression of FBP was also seen in IGROV1/Pt 0.5 and IGROV1/Pt 1. However, decreased FBP appeared to be an effect if cisplatin resistance rather than a cause of it as transfection with FBP cDNA did not cause cisplatin sensitivity
The only chemotherapy drug that IGROVCDDP was more sensitive to than IGROV-1 was 5-FU (
P-gp overexpression is rare in a model of acquired cisplatin resistance. In the IGROVCDDP cells P-gp causes taxane resistance and overrides any potential taxane sensitivity mediated by increased BRCA1 expression. Platinum resistance is multifactorial and is mediated by an increase in glutathione recycling and decreased accumulation of drug. The IGROVCDDP cells were sensitive to 5-FU and this class of chemotherapeutics warrants further preclinical research to determine if they are useful for the treatment of platinum/taxane resistant ovarian cancer.
Netherlands Cancer Institute - Prof. Jan Schellens for the IGROV-1 and IGROVCDDP cells. Dublin City University - Dr. Robert O’Connor for the P-gp antibody. Dr. Aoife Devery for optimising the P-gp westerns. Dr. Alex Eustace for the MRP2 positive control and antibody. Ms. Helena Joyce for the BCRP positive and antibody. National Cancer Institute - Drs. Michael Dean, Ding-Wu Shen, Matthew D. Hall and Mitsunori Okabe for compiling the list of 380 MDR-linked genes for the TLDA array. University of Oxford – Dr. Zoe Holloway and Prof. Anthony Monaco for the ATP7A antibody. Trinity College Dublin – Dr. Anne-Marie Byrne for the GM130 antibody.