Introduction
Ovarian cancer (OC) is the most lethal gynaecologic cancer, which accounts for 4% of all kinds of women’s cancer. The most malignant subtype of ovarian cancer is high-grade serous ovarian cancer (SOC), causing the most lethality of ovarian cancer cases [
1,
2]. So far chemotherapy is one of effective treatments for ovarian cancer. Many chemotherapeutics, such as taxol (TAX), carboplatin (CBP), doxorubicin, cyclophosphamide, etc., are widely used in clinical practice, eliminating cancer cells through inhibiting the proliferation of and promoting the death of cancer cells. For example, taxol can combine with tubulin polymers, and selectively block cell proliferation in the G2/M phase, and thus induce cytotoxicity. Carboplatin can alkylate DNA in cancer cells, resulting in the disruption of DNA structure and the death of cancer cells.
Despite advances in chemotherapy, the overall five-year survival rate for SOC is just roughly 20%, mainly due to chemoresistance, which leads to cancer recurrence and treatment failure for SOC patients [
3,
4]. Previous studies indicated that cancer chemoresistance is underlined by multiple mechanisms, including DNA repair, autophagy, drug efflux, metabolism reprogramming, epithelial-mesenchymal transition (EMT), mitochondrial alteration, etc. [
5,
6]. Molecules involved in those processes may play roles in regulating chemoresistance, and could provide potential targets for drug treatment or diagnosis, in order to improve survival rates for ovarian cancer patients.
To investigate the molecular and cellular mechanisms of ovarian cancer chemoresistance, taxol and carboplatin, two commonly used effective drugs in clinical practice, were utilized for developing chemoresistant SOC cell lines. In this study, we show here that Glutathione-S-transferases-T1 (GSTT1), a member of metabolic enzymes that catalyse the conjugation of glutathione onto endogenous and xenobiotic reactive intermediates [
7,
8], and a previously showed cancer related gene, is related to the chemoresistance of SOC cell lines. The expression level of GSTT1 is not only substantially up-regulated in taxol and carboplatin resistant SOC cell lines, but also physiologically up-regulated in the tissues from SOC patients after chemotherapy. Down-regulation of GSTT1 in SOC cells reduced cell viability and increased the sensitivity to treatments, through promoting cell death and increasing divided cells at G2/M phase. Interestingly, DNA topoisomerase 1 (Topo I), which is synergistically up-regulated in drug-resistant SOC cells, could interact with GSTT1, based on our preliminary efforts to illustrate the molecular mechanism of GSTT1’s role in chemoresistance of SOC cell lines.
Materials and methods
Ovarian cancer tissues
The study was approved by the Ethics Committee of Shanghai Cancer Center, Fudan University (Certification no. 050432–4-1212B). Prior written informed consent was obtained from all patients. Tissues were obtained from patients who had undergone an operation at Gynecologic Surgery, Fudan University Shanghai Cancer Center (Shanghai, China) between 2013 and 2015, including 40 SOC tissues (FIGO stage II-IV, median age 55 years) and 20 normal tissues (ovary tissues obtained from 20 patients who underwent ovariohysterectomy for other gynecological cancer, median age 58 years). All specimens were collected and frozen in liquid nitrogen immediately after surgery and then stored at − 80 °C until analysis. The diagnoses of all the patients were confirmed by histopathological examination.
Cell culture and reagents
Human OC cell lines SKOV3 and HO8910 were purchased from Shanghai Chinese Academy of Sciences and were cultured in RPMI-1640 medium (Gibco) containing 10% foetal bovine serum, 1% HEPES and 1% penicillin-streptomycin (Gibco). Cell culture was performed in an incubator with a CO2 concentration of 5% and a temperature of 37 °C.
Establishment of taxol / carboplatin-resistant OC cell lines (SKOV3-TAX/CBP, HO8910-TAX/CBP)
Taxol (TAX, concentration) and carboplatin (CBP) were obtained from the pharmacy department of Fudan University Shanghai Cancer Center. Taxol/carboplatin-resistant OC cell lines (SKOV3-TAX/CBP, HO8910-TAX/CBP) were established in vitro by the gradually increased concentration method with time-stepwise increment. Firstly, SKOV3 and HO8910 cells were exposed to stepwise escalating concentrations of TAX (2.5 μg/ml, 5 μg/ml, 7.5 μg/ml, 10 μg/ml and 12.5 μg/ml) for 48 h, when they grew to 70–80% confluence, to check the initial concertration. The treated cells were then washed with PBS and cultured in TAX-free RPMI-1640 that was refreshed every day. The dead cells were washed out with PBS, and fresh medium again was added daily. After the cells recovered, the initial concentration was checked as 2.5 μg/ml, and TAX was added to the cells at increased doses. While the final concentration of TAX was five-fold higher than the initial concentration, the SKOV3-TAX (12.5 μg/ml) and HO8910-TAX (12.5 μg/ml) cells were treated with low concentrations of CBP (5 μg/ml, 10 μg/ml, 15 μg/ml, 20 μg/ml and 25 μg/ml). The treatment with CBP was repeated the same as previous steps with TAX treatment, until the initial concentration was checked as 5 μg/ml and the final five-fold concentration was reached to 25 μg/ml. Then, this cyclic treatment with TAX and CBP was performed three times, as TAX and CBP respectively was reached to 10-fold, 15-fold and 20-fold higher than the initial concentration in the end (as TAX-50 μg/ml and CBP-100 μg/ml). The resistant cells were then cultured without TAX and CBP for three passages and frozen in the liquid nitrogen [
9].
Establishment of GSTT1 down-regulated transduced cells
Short hairpin RNA (shRNA) sequences (Table
1) were designed with the RNAi Designer program. The segments of nucleotides were cloned into the pLKO.1-puro vector (Addgene, MA, USA). The envelope vector pMD2.G, packing plasmid PAX2 and recombined plasmids were transfected into HEK293T cells at a ratio of 1:3:4. The virus supernatant was collected after 48 h and transduced into the OC cell lines using polybrene (8 mg/ml, Sigma, USA) for 24 h to generate shGSTT1–1 and shGSTT1–2 transduced cells. Transduced cells were screened by puromycin (1 mg/ml, Sigma-Aldrich, MO, USA) for 5 days [
10].
Table 1
Sequences of primers and targets
GSTT1 qRT-PCR forward | 5′-CCAAGCTGCACGATAGGTCAC-3′ |
GSTT1 qRT-PCR reverse | 5′-GGTATGCTACACACAGCTCCAC-3′ |
MDR1 qRT-PCR forward | 5′-GACATGACCAGGTATGCCTA-3′ |
MDR1 qRT-PCR reverse | 5′-CTTGGAGACATCATCTGTAAGTC-3′ |
Topo I qRT-PCR forward | 5′-AGGTTCCTTCTCCTCCTCCA-3′ |
Topo I qRT-PCR reverse | 5′-GCCGAGCAGTCTCGTATTTC-3′ |
ERCC1 qRT-PCR forward | 5′-CCTTATTCCGATCTACACAGAGC-3′ |
ERCC1 qRT-PCR reverse | 5′-TATTCGGCGTAGGTCTGAGGG-3’ |
Bcl2 qRT-PCR forward | 5′-TTGCCAGCCGGAACCTATG-3’ |
Bcl2 qRT-PCR reverse | 5′-CGAAGGCGACCAGCAATGATA-3’ |
Cyclin B1 qRT-PCR forward | 5′-AATAAGGCGAAGATCAACATGGC-3’ |
Cyclin B1 qRT-PCR reverse | 5′-TTTGTTACCAATGTCCCCAAGAG-3’ |
Cyclin E1 qRT-PCR forward | 5′-GCCAGCCTTGGGACAATAATG-3’ |
Cyclin E1 qRT-PCR reverse | 5′-CTTGCACGTTGAGTTTGGGT-3’ |
β-actin qRT-PCR forward | 5′- AAGGTGACAGCAGTCGGTT-3’ |
β-actin qRT-PCR reverse | 5′- TGTGTGGACTTGGGAGAGG-3’ |
shGSTT1–1 forward | 5′-CCGGCAGCACTTAAGCGATGCCTTTCTCGAGAAAGGCATCGCTTAAGTGCTGTTTTTG-3’ |
shGSTT1–1 reverse | 5′-AATTCAAAAACAGCACTTAAGCGATGCCTTTCTCGAGAAAGGCATCGCTTAAGTGCTG-3’ |
shGSTT1–2 forward | 5′-CCGGGCTTGCTTAAGACTTGCCCAACTCGAGTTGGGCAAGTCTTAAGCAAGCTTTTTG-3’ |
shGSTT1–2 reverse | 5′-AATTCAAAAAGCTTGCTTAAGACTTGCCCAACTCGAGTTGGGCAAGTCTTAAGCAAGC-3’ |
Cell cycle assay
Following incubation for 24 h, cells were harvested, washed with ice-cold PBS, and fixed with 70% alcohol overnight at 4 °C. The fixed cells were incubated with 500 μl PI (BD PharmingenTM, USA) for 15 min at room temperature in the dark and were analysed by flow cytometry. Cell distributions were assessed using ModFit LT software (Verity Software House, ME).
Cell death assay
Cells were harvested, resuspended in 0.5 ml binding buffer, and incubated with Annexin V-fluorescein isothiocyanate/PI dual stain (BD Biosciences, San Jose, CA, USA) for 20 min and determined by flow cytometry. The assays were repeated three times. Cells negative for both PI and Annexin V were considered viable cells. PI-negative and Annexin V-positive cells were considered early apoptotic cells. PI-positive and Annexin V-positive cells were considered dead cells.
Cell proliferation assay
Cell Counting Kit-8 (CCK-8; Dojindo Molecular Technologies, Inc.) was used to assess cell proliferation. The cells were seeded in 96-well plates in triplicate at a density of ~ 1 × 103 cells/well and were cultured for 1, 2, 3 and 4 days. After incubation, CCK-8 reagent was added for 2 h at 37 °C. Optical density (OD) was detected at 450 nm.
In the drug sensitivity test, cells were seeded in 96-well plates in triplicate at a density of ~ 1 × 103 cells/well for 24 h at 37 °C, and separately treated with taxol (0, 5, 10, 20, 50, 80, 100, 200 μg/ml) and carboplatin (0, 10, 40, 80, 100, 150, 200, 500 μg/ml) for 48 h at 37 °C. Then, CCK-8 reagent was added for 2 h at 37 °C. Optical density (OD) was detected at 450 nm and the IC50 was calculated.
The drug resistant cells (HO8910-TAX, HO8910-CBP, HO8910-TAX/CBP, SKOV3-TAX, SKOV3-CBP and SKOV3-TAX/CBP) and knockdown group cells (HO8910-TAX-shCON, HO8910-TAX-shGSTT1, HO8910-CBP-shCON, HO8910-CBP-shGSTT1, HO8910-TAX/CBP-shCON and HO8910-TAX/CBP-shGSTT1) were seeded in 6-well plates at a density of 1000 cells/well and incubated for 14 days. The cells were fixed with 4% paraformaldehyde at room temperature for 15 min and then stained with 0.5% crystal violet dye at room temperature for 10 min. The cell colony number was counted by microscope.
In the drug sensitivity test, the HO8910-TAX groups were treated with taxol (50 μg/ml), the HO8910-CBP groups were treated with carboplatin (100 μg/ml) and the HO8910-TAX/CBP groups were treated with taxol/carboplatin (50 μg/ml, 100 μg/ml) for 48 h at 37 °C. Then, the medium was replaced with complete culture medium and colony formation assays were performed.
Immunohistochemistry
Chemotherapy sensitive and resistant SOC tissues were detected. Pathological section was immersed in Triton for 30 min. GSTT1 was detected as the primary antibody with a rabbit antibody at a dilution of 1:500 (Abcam, MA, USA). The ABC complex antibody was incubated at room temperature for 2 h. Immunohistochemical staining was carried out by chromogenic solution.
Quantitative real-time PCR (qRT-PCR)
Total RNA was extracted from SKOV3 and HO8910 cells using Trizol reagent (Ambion, USA) according to the manufacturer’s instructions. cDNA was reverse transcribed from total RNA by a PrimeScript® RT reagent kit (TaKaRa, Japan) at 37 °C for 15 min and 85 °C for 5 s. The specific primers actin, GSTT1 and MDR1 used for qPCR were purchased from Shanghai Bio-engineering Ltd. (Table
1). PCR was performed with a SYBR Green PCR kit (Thermo Fisher Scientific, Rockford, USA) at 95 °C for 10 min, 95 °C for 15 s, 62 °C for 40 s, for 40 cycles. Gene-specific relative mRNA levels were calculated according to the standard eq. (2
-△△CT sample, 2
-△△CTcontrol).
Western-blot and co-immunoprecipitation
For western blot, total protein was collected with RIPA buffer (Biyun Tian Biotechnology Co., Ltd.). Cell lysates were resolved by SDS-PAGE, and proteins were electro-transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, USA). The PVDF membranes were blocked with 10% non-fat milk (Solarbio, Beijing, China). The primary antibodies included anti-GSTT1 (1:1000 dilution, Abcam, MA, USA), anti-BAX (1:1000 dilution, Abcam, MA, USA), anti-Bcl2 (1:1000 dilution, Abcam, MA, USA), anti-Cyclin B1 (1:1000 dilution, Abcam, MA, USA), anti-Cyclin E1 (1:1000 dilution, Abcam, MA, USA), anti-ERCC1 (1:1000 dilution, Abcam, MA, USA), anti-Topo I (1:1000 dilution, Abcam, MA, USA) and anti-MDR1 (1:2000 dilution, Arigo, MA, USA). The secondary antibody were sheep anti-rabbit and sheep anti-mouse (1:3000 dilution, Santa Cruz, Texas, USA). All proteins were stripped and re-probed with a β-actin antibody (1:3000 dilution, Abcam, MA, USA) as a loading control.
For co-immunoprecipitation, 1 mg of total protein was immunoprecipitated with diluted antibody in lysis buffer for 12 h. The antibody was eluted with 50 μL protein A and G beads. The beads were centrifuged for 5 min and the dried beads were mixed with 2X loading dye. The mixture was boiled for 10 min. The primary antibodies included anti-GSTT1 (1:40 dilution, Abcam, MA, USA) and anti-Topo I (1:40 dilution, Abcam, MA, USA). SDS-PAGE was then performed.
Immunofluorescence
Cellular immunofluorescence was performed with anti-GSTT1 (1:400 dilution, Abcam, MA, USA) and anti-Topo I (1:500 dilution, Abcam, MA, USA) as primary monoclonal antibodies and sheep anti-rabbit and anti-mouse (1:3000 dilution, Thermo Company, USA) antibody as the fluorescence-labelled secondary antibody. DAPI was purchased from Shanghai RunJie Chemical Reagent Co., Ltd. Fluorescence densities were quantified by Image J software.
Statistical analysis
Data were analyzed by GraphPad Prism statistical software. All data are shown as the means ± standard deviation (SD). The differences between multigroups were tested by ANOVA, and the differences between the two groups were tested by student’s t-tests. The P values smaller than 0.05 were considered statistically significant (* P < 0.05, * * P < 0.01, * * P < 0.001).
Discussion
The 5-year survival rate of ovarian cancer patients is less than 50%. Ovarian cancer is also known as the “top killer” of gynecological malignancies, which seriously threatens women’s health. After standardized treatment, ovarian cancer often recurs and metastasizes within 3–5 years. Glutathione S-transferases (GST) is a family of phase II metabolic enzymes with the ability to catalyse the conjugation of reduced form of glutathione (GSH) to a variety of toxic compounds including chemotherapeutic agents [
21]. Therefore, the interaction between GSTs and their substrates such as chemotherapeutic drugs for cancer treatment could affect the metabolism of these drugs, through which affecting the death and chemoresistance of cancer cells. In fact, recent studies indeed showed GSTs played roles in cancer development, progression and drug resistance [
22]. Some studies showed that the polymorphisms of a major GST family member GSTT1, were associated with some types of cancer, such as acute lymphoblastic leukemia, prostate cancer, or associated with higher tumor grade in colorectal cancer [
23‐
26]. But other research argued that GSTT1 were not associated epithelial ovarian cancer, or the survival or clinical response in colorectal cancer and esophageal cancer [
27‐
29]. Altogether, how GSTT1 plays its roles in cancer development and chemoresistance is quite controversial, and its specific roles in serous ovarian cancer remain unknown before our study.
In this study, we showed here GSTT1 was significantly upregulated in both established chemoresistant SOC cell lines and patient tissues after chemotherapeutical treatment, indicating its biological functions relevance to SOC. Up-regulation of GSTT1 was associated with SOC cell survival after chemotherapeutical treatment, and suppression of GSTT1 expression would inhibit the proliferation of SOC cells and rescue the sensitivity to taxol / carboplatin. Meanwhile, down-regulation of GSTT1 accelerated the number of resistance cell apoptosis after treatment with taxol / carboplatin, and the typical quiescence status of cell cycle arrest was transformed into division status. As expected, down-regulated of GSTT1 expression induced the expression changes of apoptosis pathway related proteins with increase of BAX and decrease of Bcl2. Additionally, the expression levels of Cyclin B1 and Cyclin E1 were both declined significantly after down-regulation of GSTT1 in resistance cells. It was documented that GST isozymes interacted with the members of mitogen-activated protein kinase (MAPK) pathways involved in cell-survival and cell death signalling mechanisms [
14,
30]. Through regulating cell cycle and cell death, GSTT1 suppression causes more vulnerability of SOC cells to chemotherapeutics, suggesting its role in enhancing SOC chemoresistance. Therefore, it can be reasonably expected that dysfunction of GSTT1 in vivo could increase sensitivity of SOC patients to chemotherapy in the future studies.
DNA topoisomerases are nuclear enzymes which modify DNA topology and have critical function in DNA replication and DNA repair [
31,
32]. Many cancer chemotherapeutics target and inactivate DNA topoisomerases so as to intervene the replication of cancer cells, through which causing cell death after treatment [
31]. To explore the molecular mechanism of GSTT1’s role in chemoresistance, it is reasonable to ask if DNA topoisomerases are potential targets of GSTT1. It was also reported that DNA topoisomerase family member Topo I activity was inhibited via induction of the MAPK signal transduction pathway. In addition, Topo I induced the phosphorylation of phospholipase Cγ1, c-Raf, ERK-1/2, and p38 MAPK, that stimulated fibroblast migration via a G protein-coupled receptor [
33,
34]. As far as the molecular mechanism of GSTT1 is concerned, the synergistic expression between GSTT1 and Topo I in SOC resistance cells suggests that there may be potential interaction between two proteins. Our data showed that GSTT1 suppression resulted in the down-regulated of Topo I, further co-immunoprecipitation experiments revealed that the interaction between GSTT1 and Topo I was existed in vitro distinctly. Thus, the results suggest that GSTT1 may involve in DNA repair during chemotherapeutical treatment with a non-enzymatic function in SOC cells. So far, how and why this interaction happens in vivo is still not known. However, there is the possibility that the cofactors in the Topo I complex could be the substrates of GSTT1. During the progression of cell cycle, the interplay between GSTT1 and its substrate in DNA repair machinery results in more quiescence cells and thus blocks cell division, through which GSTT1 could provide cell defence to drug treatment and play roles in chemoresistance.
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