PD98059 impairs the cisplatin-resistance of ovarian cancer cells by suppressing ERK pathway and epithelial mesenchymal transition process
Li Houa,1, Xiaofei Houb,1, Lijing Wanga,1, Zenghui Lia, Beibei Xina, Jing Chena, Xiaofei Gaoa and Haixia Mua,∗
aDepartment of Gynecology and Obstetrics, Yantai Yuhuangding Hospital, Yantai 264000, Shandong, China
bDepartment of Clinical Laboratory, Yantai Yuhuangding Hospital, Yantai 264000, Shandong, China
BACKGROUND: The study was aimed at investigating the role of PD98059 on impairing the cisplatin-resistance of ovarian cancer cells and figuring out the potential mechanism.
MATERIAL AND METHODS: Treated with low dose of cisplatin (DDP), DDP-resistant ovarian cancer cells were built and named as SKOV-3/DDP. The cell viabilities of ovarian cancer cell line SKOV-3 and SKOV-3/DDP were detected using MTT assay. Wound healing assay and flow cytometry were performed to detect the migratory ability and cell cycle variation of the two cells and assess the sensibility to DDP in the two cell lines. However, cotreated with DDP and PD98059, cell viability, migration and cell cycle of SKOV-3/DDP were determined again. The DDP-resistance varied a lot and the potential mechanism was studied via western blot assay.
RESULTS: Both treated with DDP, SKOV-3/DDP showed an intense resistance than SKOV-3 including stronger cell viability, larger migration area and less G1/G0 arrest, which confirmed the successfully established DDP-resistant cell line. The phos- phorylation of ERK and the activation of epithelial mesenchymal transition (EMT) process contributes to the enhanced resis- tance. PD98059, a MEK inhibitor, suppresses the ERK pathway and the EMT process of SKOV-3/DDP. Co-treated by DDP and PD98059, cell proliferation and migratory area decreased, meantime more cell were arrested in G0/G1 phase compared to simple treatment of DDP or PD98059.
CONCLUSION: PD98059 efficiently impairs the DDP-resistance of ovarian cancer cells via downregulating the ERK pathway and the EMT process.
Keywords: Ovarian cancer, cisplatin resistance, epithelial mesenchymal transition, ERK pathway
1 1. Introduction
2 Ovarian cancer is one of the most deadly female can-
3 cer diseases, with 45.6% 5-year survival [1,2]. The ab-
4 sence of specific symptoms and lack of reliable early
1These authors contributed equally to this work.
∗Corresponding author: Haixia Mu, Department of Gynecology and Obstetrics, Yantai Yuhuangding Hospital, No. 20 East Yuhuang- ding Road, Yantai 264000, Shandong, China. Tel.: +86 0535 66919
99; E-mail: [email protected].
diagnostic methods has resulted in the diagnosis of 5 70% of patients at an advanced stage . The in- 6 cipient symptoms of ovarian cancer are obscure and 7 most patients received diagnosis until entering the ad- 8 vanced stage [4–6]. Surgical treatment and platinum- 9 based chemotherapy are major therapeutic strategies 10 for ovarian cancers . Unfortunately, these therapeu- 11 tic methods seem to become less effective with the pro- 12 gression of the cancer. Moreover, the morbidity of the 13 ovarian cancer also remains a higher level due to lack 14
ISSN 1574-0153/17/$35.00 Ⓧc 2017 – IOS Press and the authors. All rights reserved
2 L. Hou et al. / PD98059 attenuates the cisplatin-resistance of ovarian cancer cells
15 of reliable predictive biomarker, ovarian cancer cells
16 metastasis and the resistance to chemotherapy [8,9].
17 Cisplatin (DDP), a platinum-based drug, is one of
18 the most valid agents of solid tumors. Platinum-based
19 drugs can cause deadly DNA damage and usually exert
20 severe cytotoxic effect on malignant cells by inducing
21 cell apoptosis. However, DDP resistance has been se-
22 rious obstruction to the recovery of patients with ovar-
23 ian cancer . In response to DDP, multiple signal-
24 ing pathways were activated and contributed to DDP-
25 resistance .
26 ERK pathway is a part of the classic MAPK (mito-
27 gen-activated protein kinase) signaling pathway, regu-
28 lating cell activities . Steinmetz et al. have reported
29 that the activation of ERK pathway will induce tu-
30 mor genesis and resistance to chemotherapeutic drugs,
31 such as cisplatin and doxorubicin [13,14]. ERK path-
32 way is increasingly activated in DDP-resistant cells,
33 and it may promote tumor cell survival in ovarian can-
34 cer [15,16]. Epithelial-mesenchymal-transition (EMT)
35 plays an important role in tumor metastasis and physi-
36 ological reaction to injury . During EMT process,
ascites of ovarian tumor) was bought from ATCC, 64 and the DDP-resistant ovarian cancer cell line SKOV- 65 3/DDP are built by our lab, applying the concentra- 66 tion of 4 mg/ml DDP stimulate to the ovarian can- 67 cer cell line SK-OV-3 repetitively. RPMI 1640 (Sigma, 68 St. Louis, MO, USA) culture medium with 10% fetal 69 bovine serum (FBS), 100 U/ml penicillin and 100 g/ml 70 streptomycin (Invitrogen, Gaithersburg, MD, USA) 71 were utilized to incubate the cells. All the cells were 72 stored in an incubator containing 5% CO2 at 37◦C. 73 The cells were divided into four groups as follows: 74 control group, DDP group (treated with 2.5 µg/mL 75 DDP for 24 h), PD98059 group (treated with 10 µM 76
PD98059 for 24 h), and PD98059 + DDP group 77
(treated with 10 µM PD98059 and 2.5 µg/ml DDP for 78
24 h). Cisplatin and PD98059 were purchased from 79
Sigma Chemical Co. (St. Louis, MO). 80
2.2. MTT assay 81
3-(4, 5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2H-tetr- 82
37 the phenotype and function of the cells will change,
38 which mainly embody in the conversion from epithe-
39 lial to mesenchymal, a loss of cellular polarization, ac-
40 quisition of mobility and invasion capacity . Com-
41 pelling evidence suggests that repressing the EMT pro-
42 cess could inhibit the proliferation, migration and in-
43 vasion of ovarian cancer cells . The work of Hasle-
44 hurst et al. strongly supported that EMT has an impor-
45 tant effect on DDP resistance in ovarian cancer .
46 PD98059 (MEK1 Inhibitor) has been shown to act
47 both in vitro and in vivo as a highly selective in-
48 hibitor of MEK1 activation and the MAP kinase cas-
49 cade [3,21]. Combination treatments with PD98059
50 and another anti-tumor reagent were widely reported to
51 enhance the cell cycle arrest and cell apoptosis [21,22].
52 In our research, we combined PD98059 and DDP for
53 promoting the inhibitory effects in DDP-resistant ovar-
54 ian cancer cells.
55 Our experimental data supported that the activation
56 of ERK pathway and EMT process evaluated the DDP
57 resistance in ovarian cancer cell line SKOV-3/DDP. As
azolium bromide (MTT) assay was utilized to ex- 83 amine cell viability. After treatment, SKOV-3 cells 84 and SKOV-3/DDP cells were collected in logarithmic 85 growth phase, and different concentrations of DDP 86 (0.00 µg/ml, 0.625 µg/ml, 1.25 µg/ml, 2.5 µg/ml, 87
5.0 µg/ml, 10.0 µg/ml) were added to the cells. Af- 88
ter that, SKOV-3 cells and SKOV-3/DDP cells were 89 placed into a 96-well plate (1 104 cells per well). 90 After incubation, cell morphology at 24 h and 48 h 91 was observed in an inverted phase contrast microscope 92 under 40 magnification (Nikon Eclipse TE2000-U, 93 Tokyo, Japan). Then 20 µl MTT reagent (5 mg/ml, 94 Sigma, St. Louis, MO, USA) was added to the cells per 95 well. After incubation for 4 h, 150 µl dimethyl sulfox- 96 ide (DMSO; Sigma, St. Louis, MO, USA) was added 97 to dissolve and crystallize the cells. The optical den- 98 sity (OD) was measured at the wavelength of 570 nm 99 under a microplate spectrophotometer. The experiment 100 was repeated three times. The inhibition rate (IR) of 101 cell growth was calculated by the following formula: 102
IR = 1 − (value in experimental groups/value in con- 103
58 an inhibitor of MEK, PD98059 impairs the DDP resis-
59 tance in SKOV-3/DDP cells, which could offer a novel
60 therapeutic strategy for overcoming DDP resistance.
trol group) × 100%.
2.3. Wound healing assay
61 2. Materials and methods
SKOV-3 cells and SKOV-3/DDP cells were seeded 106
into two 6-well plates (1 × 105 cells per well). Af-
62 2.1. Cell lines and cell culture
63 The ovarian cancer cell line SK-OV-3 (derived from
ter the cell growth reached 80%–90% confluence, the 108 single layer of the cells was scratched with a 200 µl 109 sterilized pipette tip. The scratched cells were washed 110
L. Hou et al. / PD98059 attenuates the cisplatin-resistance of ovarian cancer cells 3
with serum-free medium three times. Then the cells were incubated for 10 h at 37◦C centigrade with 5% CO2. The cells in each well were photographed and recorded. The closed area of wound was measured by
nexin V-FITC and 5 µl PI were added and cells were 159 incubated for another 15 min. The apoptosis and cell 160 cycle of the cells were then detected by flow cytometry. 161
means of the software ImageJ.
2.6. Statistical analysis
2.4. Western blot assay
Protein concentrations were determined by bicin- choninic acid (BCA) protein assay kit (Beyotime In- stitute of Biotechnology, Jiangsu, China) and pro- teins with 30–100 µg/ml concentration were sepa-
All data were analyzed using SPSS 21.0 (IBM 163 Corp., Armonk, NY, USA). The results are represented 164 as the means standard deviations (SD). One-way 165 ANOVA and two-tailed t-test were utilized to compare 166 the difference among multiple samples and the differ- 167 ence between two samples, respectively. P < 0.05 was 168
rated using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE; Bio-Rad Laborato- ries, Hercules, CA, USA). The polyvinylidene diflu- oride (PVDF; Invitrogen, Gaithersburg, MD, USA) membrane was sealed using 5% skim milk at 37 de- gree centigrade for 1 h, after which rabbit anti- pERK (Thr202/Tyr204) polyclonal antibody (1:100, ab214362, Abcam, USA), anti-ERK monoclonal anti-
considered statistically significant.
3.1. SKOV-3/DDP cells were more drug-resistant than SKOV-3 cells
body (1:1000, ab54230, Abcam, USA), anti-E-cadherin
monoclonal antibody (1:1000, ab15148, Abcam, USA), anti-Vimentin monoclonal antibody (1:1000, ab16700, Abcam, USA), anti-MEK antibody (1:1000, ab178876, Abcam, USA) and anti-β-actin monoclonal antibody (1:1000, ab8226, Abcam, USA) were applied to PVDF membrane at 4 degree centigrade overnight. Then the membrane was rinsed with tris buffer saline contain- ing 20% Tween-20 (TBST) buffer for 5 times, each time 8 min. HRP-tagged goat anti rabbit IgG (1:1000, Hangzhou Kelian Biotechnology company, China) was added and incubated at room temperature for 1–2 h and washed 5 times using TBST buffer, each time 8 min. The samples were subsequently photographed and observed under a microscope. β-actin acted as an endogenous control.
2.5. Flow cytometry (FCM) detection
FCM was employed to detect the cell cycle and apoptosis rate. We adopted Annexin V-FITC/PI dual- fluorescence apoptosis detection method to assess the apoptosis of ovarian cancer cells. The cells were placed
Treated with different concentrations of DDP for 173
24 h, SKOV-3 and SKOV-3/DDP cells were collected. 174 Cell viabilities were examined with MTT assay and 175 DDP did have a dose-dependent and time-dependent 176 response for cell growth. However, after the treatment 177 of DDP at same concentration during same time, the 178 growth inhibition rate in SKOV-3 group was signifi- 179 cantly higher than that in SKOV-3/DDP group (P < 180 0.05, Fig. 1A and B). In the following experiments, 181 both SKOV-3 and SKOV-3/DDP were previously cul- 182 tured in 2.5 µg/mL DDP for 24 h. According to FCM 183 detection, higher apoptosis rate was detected after 184 DDP addition in SKOV-3 group than SKOV-3/DDP 185 group, proving the drug resistance of SKOV-3/DDP 186 cells (Fig. 1C and D). In Fig. 1E and F, more cells were 187 captured in G0-G1 phase in SKOV-3 group, indicating 188 that DDP addition successfully blocked the mitosis and 189 proliferation while SKOV-3/DDP cells showed an in- 190 tense DDP resistance (P < 0.05). Besides, the wound 191 healing assay showed larger scratching healing area 192 in SKOV-3/DDP cells, which indicated a greater inva- 193 sion ability (Fig. 1G and H). Collective data confirmed 194 the successfully established DDP-resistant ovarian cell 195
into 12-well plates and cultured with 5 µl Annexin V
at room temperature. After that, the cells were washed and suspended in 1 binding buffer. We subsequently added propidium iodide (PI) into the plate. The cells were incubated for 30 min in the dark at 37 degree
line, the SKOV-3/DDP.
3.2. DDP/PD98059 impacted the ERK and EMT related proteins in ovarian cancer cells
centigrade. The number of cells in each phase was cal- culated by means of fluorescence activated cell sorting (FACS) flow cytometry (BD Biosciences, San Diego, CA, USA). In order to detect cell apoptosis, 5 µl An-
Expression level of related proteins in ERK path- 199 way and EMT process were detected by western blot 200 in SKOV-3 cells and SKOV-3/DDP cells. In the course 201 of EMT, E-cadherin is the biomarker of epithelial 202
4 L. Hou et al. / PD98059 attenuates the cisplatin-resistance of ovarian cancer cells
Fig. 1. (A–B) SKOV-3 cells and SKOV-3/DDP cells were detected by MTT assay at different time. Growth inhibition rate of the SKOV-3 cells and SKOV-3/DDP cells at different concentration of DDP was measured. Growth inhibition rate went up as the concentration increased. Higher inhibition rate was discovered in SKOV-3 cells compared with SKOV-3/DDP cells. Significant difference was detected between two groups, which confirmed the drug resistance of SKOV-3/DDP cells. (C–D) Proliferation was detected by flow cytometry. DDP could promote the apoptosis of SKOV-3 cells, confirming the drug resistance of SKOV-3/DDP cells. (E–F) Cell cycle was detected with flow cytometry. Cells were arrested in G0-G1 phase and G2/M phase in SKOV-3 cells, indicating that DDP blocked the proliferation of SKOV-3 cells, proving the drug resistance of SKOV-3/DDP cells (both P < 0.05). (G–H) Migration was detected by wound healing assay. Larger scratch healing area was detected in SKOV-3/DDP, revealing a worse inhibition effect in SKOV-3/DDP cells which had drug resistance. *P < 0.05, compared with SKOV-3 cells.
L. Hou et al. / PD98059 attenuates the cisplatin-resistance of ovarian cancer cells 5
Fig. 2. The expression level of ERK pathway- and EMT-related proteins were detected by western blot assay. (A) The expression of ERK pathway- and EMT-related proteins in SKOV-3/DDP cells and SKOV-3 cells before drug treatment. The expression of Vimentin in SKOV-3/DDP cells was higher. E-cadherin, MEK and p-ERK in SKOV-3/DDP cells presented a higher expression in comparison with SKOV-3 cells. (B) The expression level of ERK pathway- and EMT-related proteins in SKOV-3/DDP cells after drug treatment. The expression level of Vimentin increased, the expression levels MEK and pERK were decreased while the expression of E-cadherin displayed no significant difference in DDP group compared with the control group. The expression level of E-cadherin increased and the expression of Vimentin, MEK and pERK were decreased in PD98059 group. The expression level of E-cadherin was increased and the expression of Vimentin, MEK and pERK decreased in the group of combined the PD98059 and DDP. *P < 0.05, compared with SKOV-3 cells or control group.
cells and Vimentin is the biomarker of mesenchymal cells. MEK was the upstream protein in ERK pathway; p-ERK was the activated protein in ERK pathway. The expression of E-cadherin in SKOV-3/DDP cells was significantly lower. However, Vimentin, MEK and phosphorylation of ERK (p-ERK) in SKOV-3/DDP cells presented a considerably higher expression in comparison with SKOV-3 cells, suggesting an en- hanced ERK activation and an EMT process (all P < 0.05, Fig. 2A). No significant difference was detected in ERK expression between SKOV-3 cells and SKOV- 3/DDP cells (P > 0.05).
suggesting DDP could suppressed the ERK pathway. 222 However, using PD98059, the ERK pathway is re- 223 markably inhibited as well as the EMT process. An in- 224 creased expression level of E-cadherin, and a down- 225 regulated Vimentin, MEK and pERK were shown in 226
DDP + PD98059 group compared with the protein 227 levels in DDP group. These results indicated that the 228 PD98059 could prevent the epithelial cells transformed 229
into mesenchymal cells. However, ERK expression 230 had no significant difference in three groups (P > 231 0.05). As suggested in the figure, the combination of 232 PD98059 and DDP usage could most effectively block 233 ERK pathway and attenuated epithelial mesenchymal
After being treated with DDP, PD98059 or DDP +
PD98059, the expression of associated proteins dif-
fered a lot in SKOV-3/DDP cells (all P < 0.05, Fig. 2B). Compared with group NC, the DDP treat- ment induced a stronger EMT process which was be-
3.3. DDP promoted cell apoptosis and inhibited cell proliferation and migration
lieved to result in DDP resistance. However, the pro- tein level of MEK and p-ERK decreased in DDP group,
Cell apoptosis rate was detected in four groups, 238
and that in DDP group, PD98059 group and DDP + 239
6 L. Hou et al. / PD98059 attenuates the cisplatin-resistance of ovarian cancer cells
Fig. 3. (A–B) Cell apoptosis was confirmed by flow cytometry. Apoptosis rate of DDP group, PD98059 group and DDP + PD98059 group was significantly higher than that in control group. DDP + PD98059 group showed the highest apoptosis ratio. (C–D) Cell cycle was detected by flow cytometry. Cells were arrested in DDP group, PD98059 group and DDP + PD98059 group, indicating inhibited proliferation. (E–F) Scratch healing area was detected by wound healing assay. Migration ability of DDP group, PD98059 group and DDP + PD98059 group were lower, indicating fewer migration, especially in the joint group. *P < 0.05, compared with control group.
PD98059 group was significantly higher than the group NC (all P < 0.05). Nevertheless, there was no ob- vious difference between DDP group and PD98059 group, which indicated that DDP and PD98059 could promote apoptosis of SKOV-3/DDP cells both sepa- rately or jointly (Fig. 3A and B). Furthermore, FCM displayed that cells arrested in G0/G1 stage in DDP
PD98059 group and DDP + PD98059 group (all P < 253 0.05, Fig. 3E and F). Therefore, DDP and PD98059 254 could separately promote cell apoptosis, suppress cell 255
proliferation and migration, and the combination of 256
DDP and PD98059 displayed the highest effectiveness. 257
group, PD98059 group and DDP + PD98059 group were much more than that in control group (all P <
0.05) indicating that DDP and PD98059 could induce cell cycle arrest in SKOV-3/DDP cells individually or collectively (Fig. 3C and D). Wound healing assay displayed smaller scratch healing area in DDP group,
Ovarian cancer is one of the most lethal malignant 259 gynecological tumors. The 5-year survival in patients 260 with advanced ovarian cancer is less than 30% because 261 of the lack of effective biomarkers for diagnosis, prog- 262
L. Hou et al. / PD98059 attenuates the cisplatin-resistance of ovarian cancer cells 7
nosis, and personalized treatment . Although surgi- cal treatment and chemotherapy of ovarian cancer have improved in recent years, the prognosis of ovarian can- cer remains poor. The current standard of treatment for primary ovarian cancer is the combination of optimal cytoreductive surgery and platinum-based chemother- apy, but the platinum resistance contributes to a high rate of relapse [24,25]. Cisplatin is commonly used
cribed to activated ERK phosphorylation and EMT 311 process. Hence, we select PD98059, a MEK inhibitor, 312 to inhibit the ERK activation during the DDP treat- 313 ment. As a result, PD98059 efficiently enhanced the 314 sensibility of DDP in SKOV-3/DDP cells. The appli- 315 cation of the strategy in clinics could be challenging 316 at present, thus further in vivo experiments are essen- 317 tial. We firmly believe it could be clinical valuable for 318
for ovarian cancer treatment, but the relevant molecu- lar and cellular mechanism for cisplatin resistance re- mains unclear. Hence, it is critical to figure out the ad- ditional mechanism and overcome the cisplatin resis- tance, which can increase the total prognosis and im- prove the living qualities of female patients.
In this research, we first successfully obtained DDP- resistant SKOV-3 cells and named it SKOV-3/DDP. Activated ERK pathway and EMT process were found in SKOV-3/DDP cells. ERK pathway is a part of the
DDP-resistant female patients.
Conflict of interest
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