-
The SKOV3 cell line was kindly provided by Prof. YANG (State Key Laboratory of Biotherapy and Cancer Center/Collaborative Innovation Center for Biotherapy, West China Hospital, Sichuan University, Chengdu, China). The SK-TR30 cell line is a stable paclitaxel-resistant cell line[7]. The human ovarian cancer cell lines A2780 and A2780-TR were kindly provided by Dr. LI (Department of Obstetrics and Gynecology, Guangxi Cancer Hospital, Guangxi Medical University, Nanning, China). SKOV3 and SK-TR30 cells were grown in high glucose DMEM (DMEM/HG) medium supplemented with 10% fetal bovine serum. A2780 and A2780TR cells were grown in RPMI-1640 medium supplemented with 10% fetal bovine serum. These cells were grown at 37 °C in a humidified atmosphere containing 5% CO2. SK-TR30 and A2780-TR cells were cultured in medium containing an additional 10 nmol/L paclitaxel to maintain drug resistance and further cultured in drug-free medium for one week before follow-up experiments. Once thawed, the cell lines were routinely authenticated approximately every 6 months.
-
Antibodies against CFL-1 (#ab42824), p-CFL-1 (#ab12866), SSH-1 (#ab76943), Bc1-2 (#ab59348), caspase-3 (#ab13847), Cytochrome c (#ab90529), and GAPDH (#ab70699) were purchased from Abcam (USA). HRP-conjugated anti-mouse and anti-rabbit antibodies (#7076 and #7047, respectively) were obtained from Cell Signaling Technology (USA). Anti-SSH-1 antibody used in the immunohistochemistry (IHC) assay was supplied by ECM Biosciences LLC (#SP1711, Kentucky, USA). Paclitaxel was purchased from Cell Signaling Technology (#9807S).
-
Plasmids with ectopic overexpression of CFL-1 or p-CFL-1 [including WT (wild type), S3A (phosphodeficient cofilin mutant, Ser-3 replaced with alanine) and S3D (phosphomimetic cofilin mutant, Ser-3 replaced with an aspartate)], overexpression of human SSH-1 and non-targeting negative-control targets were supplied by Vigene Bioscience (Shandong, China). CFL-1 and SSH-1 shRNAs were also purchased from Vigene Bioscience. Plasmids were transfected in ovarian cancer cells using Lipo3000-based Transfection Reagent (System Invetrogen Inc., Palo Alto, CA, USA), according to the manufacturer’s instructions.
-
Whole-cell and cytoplasmic proteins were extracted using a ProteoJET Kit (Fermentas, USA). Mitochondrial proteins were isolated using a ReadiPrepTM Mitochondrial/Cytoplasmic Fractionation Kit (AAT Bioquest, USA). Equal amounts of proteins were separated by SDS-PAGE and transferred onto polyvinylidene fluoride membranes (Millipore, Bedford, MA, USA). The membranes were incubated with primary antibodies at 4 °C overnight, then probed with the appropriate secondary antibodies for 1 h at room temperature. GAPDH was used as the endogenous control. The bands were visualized and quantitated using the ECL Advance Detection System (Millipore, Billerica, MA, USA).
-
Total RNA was extracted using TRIzol Reagent (TAKARA, Japan) according to the manufacturer’s instructions. First-strand cDNA was generated using the PrimeScript RT reagent Kit (TAKARA). qPCR was performed using SYBR Premix Ex Taq Kits (Takara, Dalian, China) according to the manufacturer’s introductions. For real-time PCR analysis, the following primers were used: LIMK-1 forward, 5'-AGATGAGGCTGGGATGGACA-3', reverse, 5'-GTGGAGCACTCCAAGCTGTACT-3'; LIMK-2 forward, 5'-AAGCTCTACTGCCCCAAGGA-3', reverse, 5'-ATATGCAT CCCCATCCTCAA-3'; SSH-1 forward, 5'-AGTCTTCAGCACCCCCACAA-3', reverse, 5'-GTCTTCGCAACGCAGAAGGT-3'; SSH-2 forward, 5'-TCTCTGGAGCGACACGCTAA-3', reverse, 5'-CACATTGCCTGCACAGATACA-3'; SSH-3 forward, 5'-CTTGCCCCTCTGGAGTGACA-3', reverse, 5'-TGGATGGAGATGGGCTTGAA-3'; TESK-1 forward, 5'-GGCCGAAAAGATTCCTGTGT-3', reverse, 5'-CAGCTCACCCCGTAACACCT-3'; and TESK-2 forward, 5'-GCATTTGCCTTGGACTGTGA-3', reverse, 5'-CTTCTCAGCCAGGCCAAAGT-3'. The reference gene β-actin was used as the internal control for RNA quality. The 2−ΔΔCt method was used to evaluate the gene expression fold change among the groups. All experiments were performed in triplicate.
-
SKOV3 cells were transfected with plasmids and shRNA for 24 h. Then, cells were seeded into 96-well plates at a density of 4 x 103 cells/well. SKOV3 cells were exposed to paclitaxel at various concentrations (0, 2, 10, 20, 50, 100, and 200 nmol/L) for another 72 h after adhering to the plates. The Cell Counting Kit-8 (CCK-8) solution was added into each well at 0, 24, 48, and 72 h, and the cells were incubated in a 5% CO2 incubator at 37 ˚C for 2 h. Finally, the OD value was measured at 450 nm using a microplate reader (Bio-Tek, Winooski, VT, USA) and surviving fractions were calculated. Cell survival was calculated by normalizing the absorbance of untreated controls. The half-maximal inhibitory concentration (IC50) was used to reflect cell viability following paclitaxel treatment. The IC50 was obtained from the dose-response curves using Graph-Pad Prism 5.0 program (GraphPad Software, Inc.).
-
At 48 h after transfection with WT, S3D, S3A, shCFL1, and control vectors, 5 × 105 cells/well were seeded into six-well plates. The cells were scraped with a 200 µL pipette tip when they achieved 90% confluency. After washing with phosphate-buffered saline, the scraped cells were then cultured for another 24 h. Images were captured at 0 and 24 h under a light microscope. All experiments were performed in triplicate.
Cellular invasion assay was performed using Matrigel Invasion Chambers (BD Biosciences) with inserts containing an 8 µm pore-sized membrane with a thin layer of Matrigel. Cells were seeded into the upper chamber of 24-well Transwell plates at a density of 5 x 104 cells/well with serum-free medium after transfection. The bottom chamber was filled with 500 µL completed medium containing 10% fetal bovine serum. The Transwell-containing plates were incubated for 8 h. The upper chamber was washed, fixed with 70% ethanol, and stained with 0.1% crystal violet (Solarbio, C8470) for 20 min at room temperature. The cells remaining on the upper surface of the filter membrane were removed with a cotton swab. The invasive cells on the bottom of the membrane were stained and three different fields of stained cells were counted under a light microscope.
-
Prior to exposure to 10 nmol/L paclitaxel, all cells in exponential growth phase were transfected with overexpression and knockdown plasmids for 48 h. The degree of apoptosis was measured using an Annexin V Apoptosis Detection Kit (BD Biosciences, San Diego, CA, USA), according to the manufacturer’s protocol. The harvested cell suspension was incubated with annexin V for 15 min at room temperature in the dark and then detected by FACSC Calibur flow cytometry (BD Biosciences). The cell apoptosis rate was analyzed using CellQuest software (BD Biosciences).
-
For IHC analysis, we obtained formalin-fixed and paraffin-embedded tissue samples from 108 patients with FIGO stage I–IV EOC. All EOC patients involved underwent primary cytoreduction and subsequent paclitaxel-based chemotherapy in our hospital between February 2013 and June 2016. The date of the latest record retrieved was June 30, 2019. The clinical characteristics, pathologic diagnoses, and outcome information were collected from the patients' medical records and pathologic slides. The clinical and pathological features are shown in Table 1. Overall survival time (OS) was calculated as the time from surgery to the last follow-up date or death. Progression-free survival time (PFS) was calculated as the time from surgery to the time of detected recurrence or progression. Both OS and PFS were calculated from the date of pathologic diagnosis. EOC patients with PFS ≤ or > 6 months were divided into "sensitive" and "resistant" to paclitaxel-based chemotherapy groups, respectively. This study has been approved by the Beijing Hospital Medical Ethics Committee (BJ-2019BJYYEC-029-01) and registered at www.chictr.org.cn. Analysis of IHC was performed as previously described[7], with anti-p-CFL-1 (1:300) and anti-SSH-1 (1:100) antibodies. All staining was performed with HRP-conjugated anti-mouse or anti-rabbit IgG secondary antibody and visualized by DAB. Dr. Z. W. confirmed all of the pathological diagnoses, and was blinded to the clinical parameters. Staining was scored using a Foutier scale according to the percentage of positive cells and staining intensity: (−) or 0, tissue specimens without staining (0%–10%); (+) or 1, tissue specimens with weak staining (10%–25%); (++) or 2, tissue specimens with moderate staining (25%–50%) and (+++) or 3, tissue specimens with strong staining (> 50%). (−) and (+) were defined as low expression, and (++) and (+++) were defined as high expression (or overexpression)[7].
Clinicopathologic features Relative p-CFL-1 level χ2 value P value Relative SSH-1 level χ2 value P value Low, n (%) High, n (%) Low, n (%) High n (%) Age (y) < 60 26 (60.47) 17 (39.53) 1.694 0.193 23 (53.49) 20 (46.51) 0.038 0.846 ≥ 60 31 (47.69) 34 (43.31) 36 (55.38) 29 (44.62) FIGO staging I–II 20 (57.14) 15 (42.86) 0.396 0.529 17 (48.57) 18 (51.43) 0.767 0.381 III–IV 37 (50.68) 36 (49.32) 42 (57.53) 31 (42.46) Differentiation G1-2 28 (70.00) 12 (30.00) 7.605 0.020* 21 (42.50) 29 (57.50) 5.992 0.014* G3 29 (42.65) 39 (57.35) 38 (61.76) 20 (38.24) Chemotherapy response Sensitive 45 (61.64) 28 (38.36) 7.105 0.008** 34 (46.58) 39 (53.42) 5.896 0.015* Resistant 12 (34.29) 23 (65.71) 25 (71.43) 10 (28.57) Note. *P < 0.05, **P < 0.01. Table 1. Correlation between the clinicopathological features of EOC patients and the expression of SSH-1 or p-CFL-1
-
Statistical analysis was performed with SPSS 22.0 and GraphPad Prism 5.0 software. The data were expressed as mean ± SD/SEM (M ± s/sx). Student's t-test and one or two-way ANOVA were used as appropriate for experiment and treatment groups. The correlation between p-CFL-1 and SSH-1 expression in EOC patient tissues was analyzed by Spearman's χ2 test. Survival curves were generated according to the Kaplan–Meier method, and statistical analysis was performed using the log-rank test. A P-value of less than 0.05 was regarded as statistically significant.
-
This study was approved by the Ethics Committee of Beijing Hospital (2019BJYYEC-029-01) and was registered at www.chictr.org.cn (ChiCTR1900021718). Written informed consent was obtained from all participants.
-
With increased paclitaxel concentration, the levels of p-CFL-1 in A2780 and SKOV3 cells increased accordingly. However, there was no significant change in the content of total CFL-1 protein (Figure 1A). To further determine the relationship between p-CFL levels and paclitaxel treatment response, we transfected plasmids that could modify the phosphorylation state of CFL-1 into SKOV3 cells. Compared with cells transfected with the control vector, the cells overexpressing wild type CFL (WT) and p-CFL-1 (S3D) were more resistant to paclitaxel, as demonstrated by a lower cell inhibition rate [(IC50, (1.709 ± 0.086) vs. (1.068 ± 0.110) μmol/L, P = 0.003, (2.077 ± 0.189) vs. (1.068 ± 0.110) μmol/L, P = 0.002)]. By contrast, the mutant S3A with downregulated p-CFL-1 production showed higher sensitivity to drug exposure [(IC50, (0.607 ± 0.148) vs. (1.068 ± 0.110) μmol/L, P = 0.001)]. (Figure 1B, 1D-1). Similarly, when CFL-1 expression was knocked down by shRNA technology, SKOV3 cells were more sensitive to paclitaxel [(IC50, (0.484 ± 0.327) vs. (0.962 ± 0.094) μmol/L, P = 0.005)] (Figure 1C, 1D-2). Collectively, these results indicated that elevated p-CFL-1 levels are implicated in a compromised paclitaxel response.
-
We observed a remarkable difference in apoptosis rates between paclitaxel-resistant (SK-TR30 and A2780-TR) and paclitaxel-sensitive (SKOV3 and A2780) cell lines as assessed by flow cytometry with annexin V/propidium iodide (PI) double staining. Following treatment with 10 nmol/L paclitaxel for 24 h, the rates of apoptosis of paclitaxel-resistant cell lines were significantly lower than those of the corresponding sensitive cell lines [SK-TR30 vs. SKOV3, (4.11% ± 0.78%) vs. (12.07% ± 1.47%), P = 0.01; A2780-TR vs. A2780, (5.84% ± 1.23%) vs. (14.60% ± 2.50%), P = 0.01] (Figure 2A). These data suggest a putative role of apoptosis inhibition in the mechanism of paclitaxel resistance. To verify our hypothesis that phosphorylated CFL-1 inhibits apoptosis, we detected the apoptosis rate, as well as the expression of apoptosis-specific proteins in the aforementioned mutants that displayed different levels of p-CFL-1. The S3D mutant with upregulated phosphorylated CFL-1 demonstrated a significantly lower apoptosis rate than the S3A mutant that expressed dephosphorylated CFL-1 [S3D vs. S3A, (4.35% ± 0.58%) vs. (17.9% ± 2.36%), P < 0.01, Figure 2B, 2C]. The expression of apoptosis-specific proteins further confirmed the apoptotic effect of p-CFL-1 on SKOV3 cells, including anti-apoptotic regulator (Bcl-2) and apoptosis biomarkers (cytochrome c and caspase-3). Western blotting showed that expression of Bcl-2 was upregulated and expression of cytochrome c and caspase-3 was downregulated in the S3D mutant group compared with the control group (Figure 2D). Conversely, the S3A mutant and CFL-1 knockdown groups demonstrated downregulated Bcl-2 and upregulated cytochrome c and caspase-3 levels compared with the control group (Figure 2D).
Figure 2. Phospharylated-CFL-1 in the cytoplasm was associated with mitochondrial apoptosis under paclitaxel treatment.
Previous studies have shown that mitochondrial translocation of dephosphorylated CFL, the only form able to enter the mitochondria, induces apoptosis[17-20]. To further elucidate how different phosphorylation states of cofilin and their respective distribution contribute to the paclitaxel response, we compared the cellular distribution of p-CFL-1 and total CFL-1 in paclitaxel-resistant and -sensitive EOC cell lines. Although there was no significant difference in cytoplasmic CFL-1 expression between paclitaxel-resistant and -sensitive cells, a significant decrease in mitochondrial CFL-1 expression was detected in SK-TR30 and A2780-TR cells, consistent with previous studies (Figure 2E-1, 2E-2). Expression of p-CFL-1 in paclitaxel-resistant cell lines mainly localized to the cytoplasm compared with paclitaxel-sensitive cell lines. Expression of p-CFL-1 was not detected in the mitochondria of all experimental groups (Figure 2E-1, 2E-3). Therefore, we speculated that p-CFL is prevented from mitochondrial translocation to further induce apoptosis, and thus accumulates in the cytoplasm, enabling the cell to survive paclitaxel treatment.
-
We detected gene expression of four CFL phosphokinases (LIMK-1, LIMK-2, TESK-1, and TESK-2) and three CFL phosphatases (SSH-1, SSH-2, and SSH-3) in SK-TR30 and SKOV3 cells. Expression of SSH-1 and SSH-3 was downregulated, while expression of TESK-2 and LIMK-2 was upregulated in SK-TR30 cells (Figure 3A). SSH-1 was selected for further study as it exhibited the largest difference in gene expression between paclitaxel-resistant and -sensitive cells.
The expression of SSH-1 in SK-TR30 and A2780-TR cells was substantially lower than that in SKOV3 and A2780 cells, and decreased gradually with increasing paclitaxel concentration (Figure 3B, 3C). In addition, there was a negative correlation between the level of SSH-1 and the level of p-CFL-1 (Figure 3D). We also found that overexpression of SSH-1 enhanced the killing effect of paclitaxel on SKOV3 cells, while knocking down SSH-1 had a protective effect against paclitaxel, contrary to the effect of p-CFL-1 as shown by the cytotoxicity assay (Figure 3E). Altogether, these data demonstrated that paclitaxel could trigger changes in SSH-1/p-CFL-1 signaling, which made EOC cells prone to paclitaxel resistance.
-
To determine the clinical relevance of SSH-1/p-CFL-1 signaling, we investigated 108 EOC patients, including 73 patients with chemo-resistance and 35 patients with chemo-sensitivity, all of whom underwent radical surgery followed by paclitaxel-based chemotherapy. The levels of p-CFL-1 and SSH-1 protein were analyzed by immunohistochemistry staining of tissue samples. The clinical characteristics of patients and the results of IHC analysis are shown in Table 1. High expression of p-CFL-1 was negatively correlated with the differentiation of ovarian cancer, while expression of SSH-1 was positively correlated with the differentiation of EOC. As shown by immunohistochemistry staining, 65.71% (23/35) of chemo-resistant patients had high levels of p-CFL-1, and 71.43% (25/35) of chemo-resistant patients had low expression of SSH-1 (Figure 4A-1, 4B-1; Table 1). Only 38.36% (28/73) of the patients had high expression of p-CFL-1, and 46.58% (34/73) of the patients had low expression of SSH-1 in the chemo-sensitive group, which was vastly different from that in the paclitaxel-resistant group. The coexistence of a low SSH-1 level and a high p-CFL-1 level accounted for 24.66% (13/73) of chemo-sensitive patients and 60.00% (21/35) of chemo-resistant patients with significant differences (χ2= 8.355, P = 0.008) (Figure 4C-1). Spearman's correlation analysis showed that there was a strong negative correlation between the low level of SSH-1 and the high level of p-CFL-1 expression in chemo-resistant patients (r = −0.725, P < 0.001) and a moderate negative correlation in all patients (r = −0.415, P < 0.001).
Figure 4. High expression of p-CFL-1 and low expression of SSH-1 in EOC tissues were closely related with the chemotherapy response and prognosis of EOC patients.
Survival analysis was performed to evaluate the relationship between levels of p-CFL-1 and SSH-1 and clinical outcome. We assessed overall survival (OS) and progression-free survival (PFS). As indicated in Figure 4 and Table 2, a high level of p-CFL-1 was significantly correlated with poor prognosis of patients with median OS shortened by 13 months (chemo-resistant group vs. chemo-sensitive group, 34 vs. 47 months, P < 0.001) and PFS shortened by 19 months (8 vs. 26 months, P < 0.001), respectively (Figure 4A-2, 4A-3; Table 2). However, high expression of SSH-1 was markedly correlated with better prognosis of patients, with median OS shortened by 14 months (chemo-resistant group vs. chemo-sensitive group, 38 vs. 52 months, P = 0.004) and PFS shortened by 20 months (10 vs. 30 months, P = 0.04) (Figure 4B-2, 4B-3; Table 2). In addition, patients with both a low level of SSH-1 and a high level of p-CFL-1 showed significantly poorer OS and PFS compared to those with both high SSH-1 and low p-CFL-1 levels (Figure 4C-2, 4C-3; Table 2). These survival data demonstrated that a high level of p-CFL-1 and low level of SSH-1 were closely associated with the poor prognosis of EOC patients.
Chemotherapy response (n) p-CFL-1 high expression SSH-1 low expression OS PFS OS PFS Median time (m) P value Median time (m) P value Median time (m) P value Median time (m) P value Chemo-sensitivity (73) 47 < 0.001*** 26 < 0.001*** 52 0.004** 30 0.04* Chemo-resistance (35) 34 8 38 10 Note. *P ≤ 0.05, **P ≤ 0.01 and ***P ≤ 0.001.OS, overall survival; PFS, progression-free survival. Table 2. Prognosis of 108 EOC patients with high expression of p-CFL-1 or low expression of SSH-1
Phosphorylation of Cofilin-1 Enhances Paclitaxel Resistance of Epithelial Ovarian Cancer Cells by Inhibiting Apoptosis
doi: 10.3967/bes2021.063
- Received Date: 2021-03-01
- Accepted Date: 2021-05-17
-
Key words:
- Cofilin-1 /
- Slingshot-1 /
- Epithelial ovarian cancer /
- Chemo-resistance /
- Apoptosis
Abstract:
Citation: | LI Min, DONG Xu Dong, LYU Qiu Bo, ZHANG Wei, HUANG Shuai, YANG Chun Xue, CUI Di, LAI Hui Ying. Phosphorylation of Cofilin-1 Enhances Paclitaxel Resistance of Epithelial Ovarian Cancer Cells by Inhibiting Apoptosis[J]. Biomedical and Environmental Sciences, 2021, 34(6): 465-477. doi: 10.3967/bes2021.063 |