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Inactivation of transforming growth factor-β-activated kinase 1 promotes taxol efficacy in ovarian cancer cells
Biomedicine & Pharmacotherapy 84 (2016) 917–924
Available online at
ScienceDirect www.sciencedirect.com
Original article
Inactivation of transforming growth factor-b-activated kinase 1 promotes taxol efficacy in ovarian cancer cells Li Boa,b , Huchuan Cuic , Zhengxian Fangb , Tao Qunb , Caoyun Xiaa,* a
Department of Obstetrics and Gynaecology, Anhui Medical University, Hefei 230032, China Department of Obstetrics and Gynaecology, Chaohu Hospital of Anhui Medical University, Chaohu 238000, China c Clinical laboratory, Chaohu Hospital of Anhui Medical University, Chaohu 238000, China b
A R T I C L E I N F O
Article history: Received 8 July 2016 Received in revised form 22 September 2016 Accepted 27 September 2016 Keywords: TAK1 Taxol Ovarian cancer 5Z-7-oxozeaenol
A B S T R A C T
Resistance to taxol represents a major obstacle for long-term remission in ovarian cancer. Transforming Growth Factor-b-Activated Kinase 1 (TAK1) is a critical component in immune response pathway. However, the role of TAK1 in the development of chemoresistance in ovarian cancer remains unknown. Here, we showed that in vitro, taxol-resistant cells expressed higher TAK1, and the ratio of p-TAK1/TAK1 positively associated with taxol resistance in ovarian cancer cells. Inactivation of TAK1 by inhibitor 5Z-7oxozeaenol or gene knockdown sensitized taxol cytotoxicity in vitro, promoting cell apoptosis and mitosis arrest. Moreover, resistant cells were much more sensitive to the combined TAK1 inhibitor and taxol treatment than their parental counterparts. Using xenograft mouse model, we found that 5Z-7oxozeaenol significantly enhanced taxol efficacy in vivo. Thus, targeting TAK1 pathway is a promising strategy to enhance taxol response in ovarian cancer treatment. ã 2016 Published by Elsevier Masson SAS.
1. Introduction Chemotherapy resistance is a common and challenging problem in curing ovarian cancer. The high mortality of ovarian cancer is attributed to the emergence of tumor cells that are refractory to cytotoxic chemotherapy and clonally develop into recurrent tumors [1,2]. Most ovarian cancer patients require the first-line therapy, which involves the taxol chemotherapy [3]. Although patients generally benefit from this standard chemotherapy at the beginning of its course, relapse usually occurs [4,5]. As a result, a further therapy regimen is required [6]. Unfortunately, only a small percentage (10%–15%) of patients with relapse are able to achieve long-term remission [7]. Therefore, it is critical to study the molecular mechanism of taxol resistance in ovarian cancer. The transforming growth factor (TGF)-b-activated kinase 1 (TAK1) belongs to the family of mitogen-activated protein kinase kinase kinases (MAP3Ks). TAK1 can be activated in response to various stimulus like tumor necrosis factor a (TNFa),
lipopolysaccharide (LPS) and TGF-b, followed by the activation of p38, JNK, and NF-kB in different cellular contexts [8–11]. TAK1 activation requires Thr184 and Thr187 phosphorylations in its activation loop, where mutations of Thr184 and Thr187 residues reduce the kinase activity of TAK1, suggesting that autophosphorylation of these residues is necessary for TAK1 activation [12]. Although it has been reported that TAK1 is essential for innate and adaptive immune responses [13,14], its functional role in taxol therapy resistance in ovarian cancer remains elusive. Here, the purpose of this study is to investigate the role of TAK1 in taxol chemotherapy resistance in ovarian cancer and to establish a biological foundation for introducing TAK1 inhibitors to potentiate the anti-tumor effects of taxol in ovarian cancer. Therefore, the results should have translational implications to improve chemotherapy and clinical outcomes in patients with ovarian cancer. 2. Materials and methods 2.1. Cell lines and culture conditions
* Corresponding author at: Department of Obstetrics and Gynaecology, Anhui Medical University, 81 Meishan Road, Hefei 230032, China. E-mail address: [email protected] (C. Xia). http://dx.doi.org/10.1016/j.biopha.2016.09.105 0753-3322/ã 2016 Published by Elsevier Masson SAS.
Cancer cell lines including SKOV3 and OVCAR3 cells were purchased from ATCC (Rockville, MD). All cell lines used in this study were cultured in RPMI1640 medium supplemented with 10%
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fetal bovine serum. To generate chemoresistant ovarian cancer cells, SKOV3 cells were selected by continuous treatment with different concentrations of taxol (ranging from 0.2 mM to 2.0 mM). Resistant colonies were pooled and subsequently used for experiments. Chemoresistant cell lines were maintained in selective medium containing the highest concentrations of taxol.
temperature. The antibody–antigen complex was visualized by ECL-plus chemiluminescence (Amersham Pharmacia Biotech). Antibodies against TAK1 (ab171757) was purchased from Abcam. P-TAK1 (Thr 184/187) antibody (#4508), GAPDH (#5174), Cleaved PARP (#5625) and Cleaved Caspase-3 (#9664) were obtained from Cell Signaling Technology.
2.2. Cell cycle measurement
2.7. Immunohistochemistry
Cells were seeded into 6-well plates, cultured overnight, and treated with agents for the indicated time. Cells were then harvested and washed with PBS and fixed with precooled 70% ethanol at 4 C. Staining went along in PBS containing 40 mg/mL RNase A and 10 mg/mL propidium iodide in the dark for 30 min. For each sample, at least 1 104 cells were collected with FACSCalibur (BD Biosciences) and analyzed using the CELLQUEST software (BD Biosciences).
Formalin-fixed and paraffin-embedded ovarian cancer tissues were obtained from the Department of Obstetrics and Gynaecology, Chaohu Hospital of Anhui Medical University, China. The study was approved by Chaohu Hospital of Anhui Medical University Institutional Review Committee. To compare the expression levels of TAK1 and p-TAK1 (Thr184/187) in paired recurrent postchemotherapy ovarian tumors and their primary untreated tumors, we performed immunohistochemistry. All patients received standard taxol therapy for three to six cycles. Paraffin tissues were arranged in tissue microarrays to facilitate immunohistochemistry and to ensure that tissues were stained under the same conditions. The 46 samples were obtained from 23 patients diagnosed in the years 2000–2007. Their 23 primary samples were collected at diagnosis prior to chemotherapy, and the other 23 samples were post-chemotherapy specimens, collected at disease recurrence. The statistical program used was SPSS version 18. The rabbit anti-TAK1 antibody [4D10.1] (Abcam ab171757), rabbit antiphospho-TAK1 (Thr 184/187) antibody (CST #4508) were used, and their specificity was confirmed by western blotting. Sections were counterstained with hematoxylin, and immunoreactivity was scored independently by two investigators based on the percentage of positively stained cells and the intensity of staining. The percentage of positive tumor cells was calculated for each specimen and scored 0 (0% positive), 0.1 (1–9% positive), 0.5 (10–49% positive) and 1 (50% positive). The staining intensity was graded as negative (0), low (1), medium (2), or strong (3). A composite score was determined by multiplying the intensity and the positive percentage scores [15].
2.3. Annexin V-FITC apoptosis detection Cells were seeded into 6-well plates, cultured overnight and treated with different agents. Then, cells were harvested, washed, and stained by using the Annexin V-FITC Apoptosis Detection Kit (Beyotime). Fluorescence of the cells was determined immediately by flow cytometry (BD Biosciences). 2.4. Cellular viability assay Cell counting kit-8 (CCK-8) assays were performed to determine the viability of the cells treated with agents for for the indicated time. For this purpose, cells were seeded into 96-well plates, cultured overnight, and treated with agents for the indicated time. Then, 10 mL CCK-8 solution (Dojindo Laboratories) was added into 100 mL culture medium in each well according to the manufacturer's instructions, and the cells were incubated for 4 h at 37 C. The absorbance at 450 nm was measured with spectra-MAX190 (Molecular Devices). The percentage of cell proliferation inhibition was calculated as: proliferation inhibition (%) = [1 (A450treated/ A450control)] 100%. The averaged IC50values were determined with the Logit method from three independent tests. 2.5. TAK1 knockdown and TAK1 inhibitor TAK1 knockdown were also performed using two independent siRNAs targeting TAK1 (SignalSilence1 TAK1 siRNA I #6317 and II #6318, Cell Signaling Technology) and one control siRNA (SignalSilence1 Control siRNA, #6201), which were transfected using Lipofectamine RNAiMAX (Invitrogen). TAK1 inhibitor 5Z-7-oxozeaenol purchased from Sigma-Aldrich. 2.6. Western blotting Cells were lysed in SDS/Nonidet P-40 lysis buffer [1% SDS, 1% Nonidet P-40, 50 mmol/L Tris (pH 8.0), 150 mmol/L NaCl, 2 mg/mL leupeptin, 2 mg/mL aprotinin, 1 mmol/L phenylmethylsulfonyl fluoride (PMSF), 5 mmol/L NaF, and 100 mmol/L Na3VO4). The lysates were boiled for 5 min and then cleared by centrifugation at 15,000 rpm and 4 C. Protein concentration of the supernatant was determined using a BCA Protein Assay Reagent (Pierce). The lysates were further boiled for 5 min in sample buffer. Samples were then resolved by SDS-PAGE and transferred onto Immobilon-P (Millipore Corp.) sheets. The blots were first incubated in blocking buffer [5% (w/v) nonfat dry milk in Tris-buffered saline (TBS) plus 0.05% Tween 20] for 1 h. The blots were then incubated with a primary antibody for 16 h at 4 C, followed by incubation with a horseradish peroxidase–conjugated secondary antibody for 1 h at room
2.8. Xenograft mouse tumor models The study was approved by the Institutional Animal Care and Use Committee in Anhui Medical University. There were 4 experimental groups (Vehicle, Taxol, 5Z-7-oxozeaenol and Taxol + 5Z-7-oxozeaenol).5Z-7-oxozeaenol). 15 mice were included in each group. In the subcutaneous tumor model, SKOV3 cells were mixed with an equal volume of Matrigel (BD Biosciences) and injected subcutaneously (5 106 cells/injection) into female nude mice. Tumor growth was monitored by measurements of tumor diameters, and the tumor volume was calculated. Treatment with drugs started as soon as the tumor became palpable. At the end of treatment, all tumors except 4 dead mice in Taxol group and 2 dead mice in 5Z-7-oxozeaenol group were excised, weighed, and confirmed by histology. For BrdU incorporation, mice were intraperitoneally injected with 150 mg/kg and killed 2 h postinjection. Tumor cells were suspended into single cells which were detected using BrdU flowcytometry kit (BD, Catalog No. 559619). The number of total tumor cells and positively stained tumor cells were counted. 2.9. Statistical analysis The results were presented as mean s.d., and analyzed with one-way analysis of variance and Student’s t-test. All statistical analyses were performed using SPSS 22.0 software (SPSS Inc, Chicago, IL, USA). P value lower than 0.05 was denoted as statistically significant.
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Fig. 1. Expression of TAK1 and p-TAK1 in primary and recurrent ovarian cancers. (A and B) Immunohistochemistry shows levels of TAK1 (A) and p-TAK1 (Thr 184/187) (B) in recurrent samples compared to primary untreated specimens from the same patients based on paired two-tailed t-test (n = 23 pairs). Score is used to semi-quantify the expression levels. The bottom and top of the boxplots represent the first and third quartiles, the band inside the box is the median, and the lines above and below the box indicate the maximum and minimum of all of the data. (C) Representative images of TAK1 and p-TAK1 staining from matched primary and recurrent tumors. Scale bars represent 40 mm. (D and E) mRNA (D) and protein (E) expression of TAK1 and p-TAK1 in SKOV3 and its taxol-resistant cells SKOV3PR.
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3. Results
3.4. Combination of 5Z-7-oxozeaenol and taxol inhibits tumor growth of taxol-resistant ovarian tumor xenograft
3.1. Recurrent ovarian tumors present high levels of TAK1 and p-TAK1 To compare the expression levels of TAK1 in paired recurrent post-taxol therapy ovarian tumors and their primary untreated ones, we performed immunohistochemistry using two antibodies, one specific for TAK1 and the other specific for its active p-TAK1 (Thr184/187). After semi-quantitatively scored, we found that scores for TAK1 were higher in the recurrent ovarian patient samples than in the primary counterparts (Fig. 1A). In accordance, scores for active p-TAK1 (Thr184/187) were higher in recurrent tumors than in primary tumors (Fig. 1B). Representative staining for TAK1 and p-TAK1 in a pair of matched primary and recurrent ovarian tumors is shown in Fig. 1C. To further determine if TAK1 overexpression was associated with resistance of taxol, we established a taxol-resistant ovarian cancer cell line SKOV3PR. We found that SKOV3PR expressed higher levels of TAK1 and pTAK1 (Thr184/187) than the parental SKOV3 cells (Fig. 1D and E). These data suggested that TAK1 upregulation was related to resistance to taxol. 3.2. Inactivation of TAK1 enhance taxol sensitivity in ovarian cancer cells To determine if inactivation of TAK1 can enhance taxol sensitivity in ovarian cancer cells, we inactivated TAK1 using siRNAs or small molecule inhibitors. We found that TAK1 knockdown significantly enhanced taxol sensitivity as compared to control knockdown in SKOV3 and OVCAR3 cells (Fig. 2A and B). Next, we tested the effect of combining TAK1 inhibitor 5Z-7oxozeaenol with taxol in SKOV3 [16]. We observed that 5Z-7oxozeaenol suppressed TAK1 phosphorylation in a dose-dependent manner at Thr184/187 which was considered an indicator for TAK1 activation (Fig. 2C). The ability of 5Z-7-oxozeaenol to enhance taxol cytotoxicity was dose-dependent because higher 5Z-7-oxozeaenol concentrations were linked with a greater inhibitory effect (Fig. 2D). The effect of 5Z-7-oxozeaenol on taxol-induced cytotoxicity was further assessed between taxolresistant and parental cells. We found that treatment with 5Z-7oxozeaenol resulted in an apparent shift of the taxol IC50 in SKOV3PR cells compared to parental SKOV3 cells. In detail, the ratio of taxol IC50 in untreated to 5Z-7-oxozeaenol treated SKOV3 cells was 2.5, whereas in SKOV3PR it was 236 (Fig. 2E), which suggested that resistant cells were much more sensitive to the combined TAK1 inhibitor and taxol treatment than their parental counterparts.
3.3. Combination of 5Z-7-oxozeaenol and taxol induces G2/M arrest and enhances apoptosis It is known that taxol induces microtubule stabilization and leads to mitotic arrest in the late G2/M phase of the cell cycle [17]. Therefore, we quantified cell cycle distribution in SKOV3 cells treated with 5Z-7-oxozeaenol, taxol, or their combination. Based on flowcytometry analysis of SKOV3 cells using propidium iodide staining of DNA, we confirmed that taxol increased the fraction of SKOV3 cells at the G2/M phase 12 h after treatment (Fig. 3A). In contrast, SKOV3PR cells did not respond to taxol treatment alone, but arrested in G2/M phase only when the cells were co-treated with 5Z-7-oxozeaenol (Fig. 3B). In addition, the levels of apoptosis markers, cleaved PARP and cleaved caspase 3 (Fig. 3C) and Annexin V-FITC staining (Fig. 3D and E) increased when cells were cotreated with 5Z-7-oxozeaenol and taxol compared to the cells treated with vehicle control, 5Z-7-oxozeaenol, or taxol only.
In xenograft model, athymic mice bearing the SKOV3 subcutaneous tumors were treated with taxol (8 mg/kg), 5Z-7-oxozeaenol (6.5 mg/kg), or the combination. Administration of drugs lasted for 4 cycles of 3 days on/3 days off when the tumors first became palpable. We aimed to use a low dose of taxol and 5Z-7-oxozeaenol to determine the synergistic anti-tumor effect and minimize adverse effects. At the given doses, there was no significant difference in tumor weight between the control group and the groups treated with either single 5Z-7-oxozeaenol or taxol after four cycles of treatment. However, a significant decrease in tumor weight was observed in the group co-treated with 5Z-7oxozeaenol and taxol compared to the control group (p < 0.01) (Fig. 4A). Representative tumor-bearing mice and excised tumors were present in Fig. 4B. The percentage of tumor cells that incorporated bromodeoxyuridine (BrdU) was significantly lower in tumors receiving the combined 5Z-7-oxozeaenol and taxol than in other groups (p < 0.01) (Fig. 4C). 5Z-7-oxozeaenol treated tumors in mice showed a reduction of the p-TAK1 (Thr184/187)/TAK1 ratio (Fig. 4D). Body weight and splenic weight of mice in these groups were similar (Figs. 4E and F). Unfortunately, the survival rate did not show significant difference in these groups, which may be due to the limited period for observation and also the small number of mice in each group (data not show). The above results indicate that the combination of 5Z-7-oxozeaenol and taxol effectively inhibited tumor growth at doses that were highly tolerable in mice. 4. Discussion Microtubules have been well recognized as a major target for cancer treatment. The microtubule-targeting drugs include (1) vinca alkaloids (vinblastine, vincristine, vindesine, and vinorelbine) that bind to a and b-tubulin heterodimers, preventing their incorporation into microtubules leading to microtubule depolymerization; and (2) taxanes (taxol and docetaxel) that directly bind to and stabilize microtubules. In addition, to overcome the weakness of the above classic drugs, novel and potential microtubule-targeting agents are on the research and development [18–20]. Although taxanes are frequently used in treating several major types of cancer such as breast and lung carcinoma in addition to ovarian carcinoma, development of taxane resistance is common through various mechanisms [21–24]. Therefore, to overcome taxol resistance is an unmet need. TAK1 was characterized and widely accepted as a key player in pro-inflammatory cytokine signaling, including tumor necrosis factor-a (TNF-a), interleukin-1 (IL-1), and toll-like receptor (TLR) ligands, which lead to NF-kB pathway activation [9,13]. It was also suggested that the Smad-independent non-canonical TAK1 pathway is involved in TGF-b signaling pathways [10]. Interestingly, it recently reported that a novel antitubulin agent MT189 could activate JNK pathway through TAK1 [17]. In recent years it has become increasingly clear that the inflammatory and innate immune system plays a critical role in the development of anticancer treatment resistance. The deficiency of CYLD, essential ubiquitin modulators for TAK1 inactivation, enhances chemotherapy resistance in a glioblastoma model [25]. TAK1 is becoming a new focus in studies of anticancer therapy resistance. TAK1 is upstream of transcription factors Jun and ATF family members. These TFs play roles in drug resistance and anti-apoptosis [26,27]. Also, NF-kB is activated by TAK1-mediated signals on a pathway involving IKKs [28]. NF-kB plays roles in canonical tumorpromoting pathways through strongly transcribing critical genes and miRNAs which involve in drug resistance and anti-apoptosis [29,30].
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Fig. 2. TAK1 knockdown or TAK1 inhibitor reduces ovarian cancer Cell growth. (A and B) Immunoblot shows TAK1 knockdown efficiency 48 h after transfection and GAPDH is the loading control. Cell viability assay in SKOV3 (A) and OVCAR3 cells (B) were treated with taxol at indicated concentrations for 48 h. (C) Western blot analysis of p-TAK1 and TAK1 expression in SKOV3 cells after 48 h 5Z-7-oxozeaenol treatment. (D) Cell viability assay in SKOV3 cells treated with taxol and 5Z-7-oxozeaenol at indicated concentrations for 48 h. (E) Dose-response curves showing the effect of addition of 5Z-7-oxozeaenol to taxol in taxol-resistant SKOV3PR cells as compared to naive SKOV3 cells. Results are shown as Mean SD.
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Fig. 3. Combination of 5Z-7-oxozeaenol and taxol induces G2/M arrest and enhances apoptosis. (A and B) Percentages of cells in different stages of the cell cycle status after 12 h treatment with 2.5 mM 5Z-7-oxozeaenol, 100 nM taxol or the combination in SKOV3 (A) and SKOV3PR cells (B) were determined by staining with propidium iodide. (C–E) Apoptotic activity as indicated by cleaved PARP and cleaved caspase 3 (C) and the representative Annexin V-FITC staining (D) and its quantity (E) in SKOV3PR cells treated with 2.5 mM 5Z-7-oxozeaenol, 100 nM taxol or the combination for 16 h or 24 h. Results are shown as Mean SD.
Given the high expression level of TAK1 in patients with taxoltherapy relapse, we hypothesized that inhibition of TAK1 activation will potentiate the efficacy of taxol. Herein, using SKOV3 and OVCAR3 ovarian cell lines, we provide convincing evidence that
synthetic inhibition of TAK1 activity greatly enhances taxolinduced cytotoxicity in ovarian cells both in vitro and in vivo. Furthermore, using a chemoresistant ovarian cell line SKOV3PR, we showed that TAK1 inhibition could overcome established
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Fig. 4. Combination of taxol and 5Z-7-oxozeaenol decreases tumor growth in mice. (A–C) Tumor weights (A), representative tumor-bearing mice and excised tumors (B) and BrdU representative staining and quantity (C) of the SKOV3 subcutaneous tumor model treated as indicated. (D) The representative immunoblot and the quantitative analysis of p-TAK1 (Thr184/187)/TAK1 ratio in tumors. * p < 0.05; ** p < 0.01. (E and F) Animal body weight (D) and spleen weight (E) of the SKOV3 subcutaneous tumor model treated as indicated.
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chemoresistance in ovarian cells. In our studies, 5Z-7-oxozeaenol inhibited TAK1 activation and increased the cytotoxic effect of taxol in ovarian cells. In conclusion, this study provides pre-clinical evidence that inhibiting TAK1 sensitizes tumor cells to taxol, especially for those that have developed taxol resistance. Our results suggest that combination of TAK1 inhibitor 5Z-7-oxozeaenol with chemotherapeutic drugs might be a potential therapeutic option for the treatment of relapsed ovarian cancer patients. Declaration of interest All authors declare they have no conflicts of interest. Acknowledgements This study was supported by grants from Anhui Medical University, Hefei, China and the Hospital of Anhui Medical University, Chaohu, China to C. X. References [1] M. Cooley, et al., Molecular determinants of chemotherapy resistance in ovarian cancer, Pharmacogenomics 16 (16) (2015) 1763–1767. [2] D. Holmes, Ovarian cancer: beyond resistance, Nature 527 (7579) (2015) S217. [3] A. Kumar, P.J. Hoskins, A.V. Tinker, Dose-dense paclitaxel in advanced ovarian cancer, Clin. Oncol. (R. Coll. Radiol.) 27 (1) (2015) 40–47. [4] S.M. Lenhard, et al., Relapse and survival in early-stage ovarian cancer, Arch. Gynecol. Obstet. 280 (1) (2009) 71–77. [5] P.J. Hoskins, N. Le, Identifying patients unlikely to benefit from further chemotherapy: a descriptive study of outcome at each relapse in ovarian cancer, Gynecol. Oncol. 97 (3) (2005) 862–869. [6] S. Pignata, et al., Pazopanib plus weekly paclitaxel versus weekly paclitaxel alone for platinum-resistant or platinum-refractory advanced ovarian cancer (MITO 11): a randomised, open-label, phase 2 trial, Lancet Oncol. 16 (5) (2015) 561–568. [7] N. Li, et al., Copy number changes of 4-gene set may predict early relapse in advanced epithelial ovarian cancer after initial platinum-paclitaxel chemotherapy, Am. J. Cancer Res. 4 (3) (2014) 285–292. [8] S. Morioka, et al., TAK1 kinase switches cell fate from apoptosis to necrosis following TNF stimulation, J. Cell Biol. 204 (4) (2014) 607–623. [9] A. He, et al., TLR4-MyD88-TRAF6-TAK1 complex-mediated NF-kappaB activation contribute to the anti-inflammatory effect of V8 in LPS-induced human cervical cancer SiHa cells, Inflammation 39 (1) (2016) 172–181. [10] Z. Dvashi, et al., TGF-beta1 induced transdifferentiation of rpe cells is mediated by TAK1, PLoS One 10 (4) (2015) e0122229. [11] X. Cang, et al., Mapping the functional binding sites of cholesterol in beta2adrenergic receptor by long-time molecular dynamics simulations, J. Phys. Chem. B 117 (4) (2013) 1085–1094.
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Available online at
ScienceDirect www.sciencedirect.com
Original article
Inactivation of transforming growth factor-b-activated kinase 1 promotes taxol efficacy in ovarian cancer cells Li Boa,b , Huchuan Cuic , Zhengxian Fangb , Tao Qunb , Caoyun Xiaa,* a
Department of Obstetrics and Gynaecology, Anhui Medical University, Hefei 230032, China Department of Obstetrics and Gynaecology, Chaohu Hospital of Anhui Medical University, Chaohu 238000, China c Clinical laboratory, Chaohu Hospital of Anhui Medical University, Chaohu 238000, China b
A R T I C L E I N F O
Article history: Received 8 July 2016 Received in revised form 22 September 2016 Accepted 27 September 2016 Keywords: TAK1 Taxol Ovarian cancer 5Z-7-oxozeaenol
A B S T R A C T
Resistance to taxol represents a major obstacle for long-term remission in ovarian cancer. Transforming Growth Factor-b-Activated Kinase 1 (TAK1) is a critical component in immune response pathway. However, the role of TAK1 in the development of chemoresistance in ovarian cancer remains unknown. Here, we showed that in vitro, taxol-resistant cells expressed higher TAK1, and the ratio of p-TAK1/TAK1 positively associated with taxol resistance in ovarian cancer cells. Inactivation of TAK1 by inhibitor 5Z-7oxozeaenol or gene knockdown sensitized taxol cytotoxicity in vitro, promoting cell apoptosis and mitosis arrest. Moreover, resistant cells were much more sensitive to the combined TAK1 inhibitor and taxol treatment than their parental counterparts. Using xenograft mouse model, we found that 5Z-7oxozeaenol significantly enhanced taxol efficacy in vivo. Thus, targeting TAK1 pathway is a promising strategy to enhance taxol response in ovarian cancer treatment. ã 2016 Published by Elsevier Masson SAS.
1. Introduction Chemotherapy resistance is a common and challenging problem in curing ovarian cancer. The high mortality of ovarian cancer is attributed to the emergence of tumor cells that are refractory to cytotoxic chemotherapy and clonally develop into recurrent tumors [1,2]. Most ovarian cancer patients require the first-line therapy, which involves the taxol chemotherapy [3]. Although patients generally benefit from this standard chemotherapy at the beginning of its course, relapse usually occurs [4,5]. As a result, a further therapy regimen is required [6]. Unfortunately, only a small percentage (10%–15%) of patients with relapse are able to achieve long-term remission [7]. Therefore, it is critical to study the molecular mechanism of taxol resistance in ovarian cancer. The transforming growth factor (TGF)-b-activated kinase 1 (TAK1) belongs to the family of mitogen-activated protein kinase kinase kinases (MAP3Ks). TAK1 can be activated in response to various stimulus like tumor necrosis factor a (TNFa),
lipopolysaccharide (LPS) and TGF-b, followed by the activation of p38, JNK, and NF-kB in different cellular contexts [8–11]. TAK1 activation requires Thr184 and Thr187 phosphorylations in its activation loop, where mutations of Thr184 and Thr187 residues reduce the kinase activity of TAK1, suggesting that autophosphorylation of these residues is necessary for TAK1 activation [12]. Although it has been reported that TAK1 is essential for innate and adaptive immune responses [13,14], its functional role in taxol therapy resistance in ovarian cancer remains elusive. Here, the purpose of this study is to investigate the role of TAK1 in taxol chemotherapy resistance in ovarian cancer and to establish a biological foundation for introducing TAK1 inhibitors to potentiate the anti-tumor effects of taxol in ovarian cancer. Therefore, the results should have translational implications to improve chemotherapy and clinical outcomes in patients with ovarian cancer. 2. Materials and methods 2.1. Cell lines and culture conditions
* Corresponding author at: Department of Obstetrics and Gynaecology, Anhui Medical University, 81 Meishan Road, Hefei 230032, China. E-mail address: [email protected] (C. Xia). http://dx.doi.org/10.1016/j.biopha.2016.09.105 0753-3322/ã 2016 Published by Elsevier Masson SAS.
Cancer cell lines including SKOV3 and OVCAR3 cells were purchased from ATCC (Rockville, MD). All cell lines used in this study were cultured in RPMI1640 medium supplemented with 10%
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fetal bovine serum. To generate chemoresistant ovarian cancer cells, SKOV3 cells were selected by continuous treatment with different concentrations of taxol (ranging from 0.2 mM to 2.0 mM). Resistant colonies were pooled and subsequently used for experiments. Chemoresistant cell lines were maintained in selective medium containing the highest concentrations of taxol.
temperature. The antibody–antigen complex was visualized by ECL-plus chemiluminescence (Amersham Pharmacia Biotech). Antibodies against TAK1 (ab171757) was purchased from Abcam. P-TAK1 (Thr 184/187) antibody (#4508), GAPDH (#5174), Cleaved PARP (#5625) and Cleaved Caspase-3 (#9664) were obtained from Cell Signaling Technology.
2.2. Cell cycle measurement
2.7. Immunohistochemistry
Cells were seeded into 6-well plates, cultured overnight, and treated with agents for the indicated time. Cells were then harvested and washed with PBS and fixed with precooled 70% ethanol at 4 C. Staining went along in PBS containing 40 mg/mL RNase A and 10 mg/mL propidium iodide in the dark for 30 min. For each sample, at least 1 104 cells were collected with FACSCalibur (BD Biosciences) and analyzed using the CELLQUEST software (BD Biosciences).
Formalin-fixed and paraffin-embedded ovarian cancer tissues were obtained from the Department of Obstetrics and Gynaecology, Chaohu Hospital of Anhui Medical University, China. The study was approved by Chaohu Hospital of Anhui Medical University Institutional Review Committee. To compare the expression levels of TAK1 and p-TAK1 (Thr184/187) in paired recurrent postchemotherapy ovarian tumors and their primary untreated tumors, we performed immunohistochemistry. All patients received standard taxol therapy for three to six cycles. Paraffin tissues were arranged in tissue microarrays to facilitate immunohistochemistry and to ensure that tissues were stained under the same conditions. The 46 samples were obtained from 23 patients diagnosed in the years 2000–2007. Their 23 primary samples were collected at diagnosis prior to chemotherapy, and the other 23 samples were post-chemotherapy specimens, collected at disease recurrence. The statistical program used was SPSS version 18. The rabbit anti-TAK1 antibody [4D10.1] (Abcam ab171757), rabbit antiphospho-TAK1 (Thr 184/187) antibody (CST #4508) were used, and their specificity was confirmed by western blotting. Sections were counterstained with hematoxylin, and immunoreactivity was scored independently by two investigators based on the percentage of positively stained cells and the intensity of staining. The percentage of positive tumor cells was calculated for each specimen and scored 0 (0% positive), 0.1 (1–9% positive), 0.5 (10–49% positive) and 1 (50% positive). The staining intensity was graded as negative (0), low (1), medium (2), or strong (3). A composite score was determined by multiplying the intensity and the positive percentage scores [15].
2.3. Annexin V-FITC apoptosis detection Cells were seeded into 6-well plates, cultured overnight and treated with different agents. Then, cells were harvested, washed, and stained by using the Annexin V-FITC Apoptosis Detection Kit (Beyotime). Fluorescence of the cells was determined immediately by flow cytometry (BD Biosciences). 2.4. Cellular viability assay Cell counting kit-8 (CCK-8) assays were performed to determine the viability of the cells treated with agents for for the indicated time. For this purpose, cells were seeded into 96-well plates, cultured overnight, and treated with agents for the indicated time. Then, 10 mL CCK-8 solution (Dojindo Laboratories) was added into 100 mL culture medium in each well according to the manufacturer's instructions, and the cells were incubated for 4 h at 37 C. The absorbance at 450 nm was measured with spectra-MAX190 (Molecular Devices). The percentage of cell proliferation inhibition was calculated as: proliferation inhibition (%) = [1 (A450treated/ A450control)] 100%. The averaged IC50values were determined with the Logit method from three independent tests. 2.5. TAK1 knockdown and TAK1 inhibitor TAK1 knockdown were also performed using two independent siRNAs targeting TAK1 (SignalSilence1 TAK1 siRNA I #6317 and II #6318, Cell Signaling Technology) and one control siRNA (SignalSilence1 Control siRNA, #6201), which were transfected using Lipofectamine RNAiMAX (Invitrogen). TAK1 inhibitor 5Z-7-oxozeaenol purchased from Sigma-Aldrich. 2.6. Western blotting Cells were lysed in SDS/Nonidet P-40 lysis buffer [1% SDS, 1% Nonidet P-40, 50 mmol/L Tris (pH 8.0), 150 mmol/L NaCl, 2 mg/mL leupeptin, 2 mg/mL aprotinin, 1 mmol/L phenylmethylsulfonyl fluoride (PMSF), 5 mmol/L NaF, and 100 mmol/L Na3VO4). The lysates were boiled for 5 min and then cleared by centrifugation at 15,000 rpm and 4 C. Protein concentration of the supernatant was determined using a BCA Protein Assay Reagent (Pierce). The lysates were further boiled for 5 min in sample buffer. Samples were then resolved by SDS-PAGE and transferred onto Immobilon-P (Millipore Corp.) sheets. The blots were first incubated in blocking buffer [5% (w/v) nonfat dry milk in Tris-buffered saline (TBS) plus 0.05% Tween 20] for 1 h. The blots were then incubated with a primary antibody for 16 h at 4 C, followed by incubation with a horseradish peroxidase–conjugated secondary antibody for 1 h at room
2.8. Xenograft mouse tumor models The study was approved by the Institutional Animal Care and Use Committee in Anhui Medical University. There were 4 experimental groups (Vehicle, Taxol, 5Z-7-oxozeaenol and Taxol + 5Z-7-oxozeaenol).5Z-7-oxozeaenol). 15 mice were included in each group. In the subcutaneous tumor model, SKOV3 cells were mixed with an equal volume of Matrigel (BD Biosciences) and injected subcutaneously (5 106 cells/injection) into female nude mice. Tumor growth was monitored by measurements of tumor diameters, and the tumor volume was calculated. Treatment with drugs started as soon as the tumor became palpable. At the end of treatment, all tumors except 4 dead mice in Taxol group and 2 dead mice in 5Z-7-oxozeaenol group were excised, weighed, and confirmed by histology. For BrdU incorporation, mice were intraperitoneally injected with 150 mg/kg and killed 2 h postinjection. Tumor cells were suspended into single cells which were detected using BrdU flowcytometry kit (BD, Catalog No. 559619). The number of total tumor cells and positively stained tumor cells were counted. 2.9. Statistical analysis The results were presented as mean s.d., and analyzed with one-way analysis of variance and Student’s t-test. All statistical analyses were performed using SPSS 22.0 software (SPSS Inc, Chicago, IL, USA). P value lower than 0.05 was denoted as statistically significant.
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Fig. 1. Expression of TAK1 and p-TAK1 in primary and recurrent ovarian cancers. (A and B) Immunohistochemistry shows levels of TAK1 (A) and p-TAK1 (Thr 184/187) (B) in recurrent samples compared to primary untreated specimens from the same patients based on paired two-tailed t-test (n = 23 pairs). Score is used to semi-quantify the expression levels. The bottom and top of the boxplots represent the first and third quartiles, the band inside the box is the median, and the lines above and below the box indicate the maximum and minimum of all of the data. (C) Representative images of TAK1 and p-TAK1 staining from matched primary and recurrent tumors. Scale bars represent 40 mm. (D and E) mRNA (D) and protein (E) expression of TAK1 and p-TAK1 in SKOV3 and its taxol-resistant cells SKOV3PR.
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3. Results
3.4. Combination of 5Z-7-oxozeaenol and taxol inhibits tumor growth of taxol-resistant ovarian tumor xenograft
3.1. Recurrent ovarian tumors present high levels of TAK1 and p-TAK1 To compare the expression levels of TAK1 in paired recurrent post-taxol therapy ovarian tumors and their primary untreated ones, we performed immunohistochemistry using two antibodies, one specific for TAK1 and the other specific for its active p-TAK1 (Thr184/187). After semi-quantitatively scored, we found that scores for TAK1 were higher in the recurrent ovarian patient samples than in the primary counterparts (Fig. 1A). In accordance, scores for active p-TAK1 (Thr184/187) were higher in recurrent tumors than in primary tumors (Fig. 1B). Representative staining for TAK1 and p-TAK1 in a pair of matched primary and recurrent ovarian tumors is shown in Fig. 1C. To further determine if TAK1 overexpression was associated with resistance of taxol, we established a taxol-resistant ovarian cancer cell line SKOV3PR. We found that SKOV3PR expressed higher levels of TAK1 and pTAK1 (Thr184/187) than the parental SKOV3 cells (Fig. 1D and E). These data suggested that TAK1 upregulation was related to resistance to taxol. 3.2. Inactivation of TAK1 enhance taxol sensitivity in ovarian cancer cells To determine if inactivation of TAK1 can enhance taxol sensitivity in ovarian cancer cells, we inactivated TAK1 using siRNAs or small molecule inhibitors. We found that TAK1 knockdown significantly enhanced taxol sensitivity as compared to control knockdown in SKOV3 and OVCAR3 cells (Fig. 2A and B). Next, we tested the effect of combining TAK1 inhibitor 5Z-7oxozeaenol with taxol in SKOV3 [16]. We observed that 5Z-7oxozeaenol suppressed TAK1 phosphorylation in a dose-dependent manner at Thr184/187 which was considered an indicator for TAK1 activation (Fig. 2C). The ability of 5Z-7-oxozeaenol to enhance taxol cytotoxicity was dose-dependent because higher 5Z-7-oxozeaenol concentrations were linked with a greater inhibitory effect (Fig. 2D). The effect of 5Z-7-oxozeaenol on taxol-induced cytotoxicity was further assessed between taxolresistant and parental cells. We found that treatment with 5Z-7oxozeaenol resulted in an apparent shift of the taxol IC50 in SKOV3PR cells compared to parental SKOV3 cells. In detail, the ratio of taxol IC50 in untreated to 5Z-7-oxozeaenol treated SKOV3 cells was 2.5, whereas in SKOV3PR it was 236 (Fig. 2E), which suggested that resistant cells were much more sensitive to the combined TAK1 inhibitor and taxol treatment than their parental counterparts.
3.3. Combination of 5Z-7-oxozeaenol and taxol induces G2/M arrest and enhances apoptosis It is known that taxol induces microtubule stabilization and leads to mitotic arrest in the late G2/M phase of the cell cycle [17]. Therefore, we quantified cell cycle distribution in SKOV3 cells treated with 5Z-7-oxozeaenol, taxol, or their combination. Based on flowcytometry analysis of SKOV3 cells using propidium iodide staining of DNA, we confirmed that taxol increased the fraction of SKOV3 cells at the G2/M phase 12 h after treatment (Fig. 3A). In contrast, SKOV3PR cells did not respond to taxol treatment alone, but arrested in G2/M phase only when the cells were co-treated with 5Z-7-oxozeaenol (Fig. 3B). In addition, the levels of apoptosis markers, cleaved PARP and cleaved caspase 3 (Fig. 3C) and Annexin V-FITC staining (Fig. 3D and E) increased when cells were cotreated with 5Z-7-oxozeaenol and taxol compared to the cells treated with vehicle control, 5Z-7-oxozeaenol, or taxol only.
In xenograft model, athymic mice bearing the SKOV3 subcutaneous tumors were treated with taxol (8 mg/kg), 5Z-7-oxozeaenol (6.5 mg/kg), or the combination. Administration of drugs lasted for 4 cycles of 3 days on/3 days off when the tumors first became palpable. We aimed to use a low dose of taxol and 5Z-7-oxozeaenol to determine the synergistic anti-tumor effect and minimize adverse effects. At the given doses, there was no significant difference in tumor weight between the control group and the groups treated with either single 5Z-7-oxozeaenol or taxol after four cycles of treatment. However, a significant decrease in tumor weight was observed in the group co-treated with 5Z-7oxozeaenol and taxol compared to the control group (p < 0.01) (Fig. 4A). Representative tumor-bearing mice and excised tumors were present in Fig. 4B. The percentage of tumor cells that incorporated bromodeoxyuridine (BrdU) was significantly lower in tumors receiving the combined 5Z-7-oxozeaenol and taxol than in other groups (p < 0.01) (Fig. 4C). 5Z-7-oxozeaenol treated tumors in mice showed a reduction of the p-TAK1 (Thr184/187)/TAK1 ratio (Fig. 4D). Body weight and splenic weight of mice in these groups were similar (Figs. 4E and F). Unfortunately, the survival rate did not show significant difference in these groups, which may be due to the limited period for observation and also the small number of mice in each group (data not show). The above results indicate that the combination of 5Z-7-oxozeaenol and taxol effectively inhibited tumor growth at doses that were highly tolerable in mice. 4. Discussion Microtubules have been well recognized as a major target for cancer treatment. The microtubule-targeting drugs include (1) vinca alkaloids (vinblastine, vincristine, vindesine, and vinorelbine) that bind to a and b-tubulin heterodimers, preventing their incorporation into microtubules leading to microtubule depolymerization; and (2) taxanes (taxol and docetaxel) that directly bind to and stabilize microtubules. In addition, to overcome the weakness of the above classic drugs, novel and potential microtubule-targeting agents are on the research and development [18–20]. Although taxanes are frequently used in treating several major types of cancer such as breast and lung carcinoma in addition to ovarian carcinoma, development of taxane resistance is common through various mechanisms [21–24]. Therefore, to overcome taxol resistance is an unmet need. TAK1 was characterized and widely accepted as a key player in pro-inflammatory cytokine signaling, including tumor necrosis factor-a (TNF-a), interleukin-1 (IL-1), and toll-like receptor (TLR) ligands, which lead to NF-kB pathway activation [9,13]. It was also suggested that the Smad-independent non-canonical TAK1 pathway is involved in TGF-b signaling pathways [10]. Interestingly, it recently reported that a novel antitubulin agent MT189 could activate JNK pathway through TAK1 [17]. In recent years it has become increasingly clear that the inflammatory and innate immune system plays a critical role in the development of anticancer treatment resistance. The deficiency of CYLD, essential ubiquitin modulators for TAK1 inactivation, enhances chemotherapy resistance in a glioblastoma model [25]. TAK1 is becoming a new focus in studies of anticancer therapy resistance. TAK1 is upstream of transcription factors Jun and ATF family members. These TFs play roles in drug resistance and anti-apoptosis [26,27]. Also, NF-kB is activated by TAK1-mediated signals on a pathway involving IKKs [28]. NF-kB plays roles in canonical tumorpromoting pathways through strongly transcribing critical genes and miRNAs which involve in drug resistance and anti-apoptosis [29,30].
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Fig. 2. TAK1 knockdown or TAK1 inhibitor reduces ovarian cancer Cell growth. (A and B) Immunoblot shows TAK1 knockdown efficiency 48 h after transfection and GAPDH is the loading control. Cell viability assay in SKOV3 (A) and OVCAR3 cells (B) were treated with taxol at indicated concentrations for 48 h. (C) Western blot analysis of p-TAK1 and TAK1 expression in SKOV3 cells after 48 h 5Z-7-oxozeaenol treatment. (D) Cell viability assay in SKOV3 cells treated with taxol and 5Z-7-oxozeaenol at indicated concentrations for 48 h. (E) Dose-response curves showing the effect of addition of 5Z-7-oxozeaenol to taxol in taxol-resistant SKOV3PR cells as compared to naive SKOV3 cells. Results are shown as Mean SD.
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Fig. 3. Combination of 5Z-7-oxozeaenol and taxol induces G2/M arrest and enhances apoptosis. (A and B) Percentages of cells in different stages of the cell cycle status after 12 h treatment with 2.5 mM 5Z-7-oxozeaenol, 100 nM taxol or the combination in SKOV3 (A) and SKOV3PR cells (B) were determined by staining with propidium iodide. (C–E) Apoptotic activity as indicated by cleaved PARP and cleaved caspase 3 (C) and the representative Annexin V-FITC staining (D) and its quantity (E) in SKOV3PR cells treated with 2.5 mM 5Z-7-oxozeaenol, 100 nM taxol or the combination for 16 h or 24 h. Results are shown as Mean SD.
Given the high expression level of TAK1 in patients with taxoltherapy relapse, we hypothesized that inhibition of TAK1 activation will potentiate the efficacy of taxol. Herein, using SKOV3 and OVCAR3 ovarian cell lines, we provide convincing evidence that
synthetic inhibition of TAK1 activity greatly enhances taxolinduced cytotoxicity in ovarian cells both in vitro and in vivo. Furthermore, using a chemoresistant ovarian cell line SKOV3PR, we showed that TAK1 inhibition could overcome established
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Fig. 4. Combination of taxol and 5Z-7-oxozeaenol decreases tumor growth in mice. (A–C) Tumor weights (A), representative tumor-bearing mice and excised tumors (B) and BrdU representative staining and quantity (C) of the SKOV3 subcutaneous tumor model treated as indicated. (D) The representative immunoblot and the quantitative analysis of p-TAK1 (Thr184/187)/TAK1 ratio in tumors. * p < 0.05; ** p < 0.01. (E and F) Animal body weight (D) and spleen weight (E) of the SKOV3 subcutaneous tumor model treated as indicated.
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chemoresistance in ovarian cells. In our studies, 5Z-7-oxozeaenol inhibited TAK1 activation and increased the cytotoxic effect of taxol in ovarian cells. In conclusion, this study provides pre-clinical evidence that inhibiting TAK1 sensitizes tumor cells to taxol, especially for those that have developed taxol resistance. Our results suggest that combination of TAK1 inhibitor 5Z-7-oxozeaenol with chemotherapeutic drugs might be a potential therapeutic option for the treatment of relapsed ovarian cancer patients. Declaration of interest All authors declare they have no conflicts of interest. Acknowledgements This study was supported by grants from Anhui Medical University, Hefei, China and the Hospital of Anhui Medical University, Chaohu, China to C. X. References [1] M. Cooley, et al., Molecular determinants of chemotherapy resistance in ovarian cancer, Pharmacogenomics 16 (16) (2015) 1763–1767. [2] D. Holmes, Ovarian cancer: beyond resistance, Nature 527 (7579) (2015) S217. [3] A. Kumar, P.J. Hoskins, A.V. Tinker, Dose-dense paclitaxel in advanced ovarian cancer, Clin. Oncol. (R. Coll. Radiol.) 27 (1) (2015) 40–47. [4] S.M. Lenhard, et al., Relapse and survival in early-stage ovarian cancer, Arch. Gynecol. Obstet. 280 (1) (2009) 71–77. [5] P.J. Hoskins, N. Le, Identifying patients unlikely to benefit from further chemotherapy: a descriptive study of outcome at each relapse in ovarian cancer, Gynecol. Oncol. 97 (3) (2005) 862–869. [6] S. Pignata, et al., Pazopanib plus weekly paclitaxel versus weekly paclitaxel alone for platinum-resistant or platinum-refractory advanced ovarian cancer (MITO 11): a randomised, open-label, phase 2 trial, Lancet Oncol. 16 (5) (2015) 561–568. [7] N. Li, et al., Copy number changes of 4-gene set may predict early relapse in advanced epithelial ovarian cancer after initial platinum-paclitaxel chemotherapy, Am. J. Cancer Res. 4 (3) (2014) 285–292. [8] S. Morioka, et al., TAK1 kinase switches cell fate from apoptosis to necrosis following TNF stimulation, J. Cell Biol. 204 (4) (2014) 607–623. [9] A. He, et al., TLR4-MyD88-TRAF6-TAK1 complex-mediated NF-kappaB activation contribute to the anti-inflammatory effect of V8 in LPS-induced human cervical cancer SiHa cells, Inflammation 39 (1) (2016) 172–181. [10] Z. Dvashi, et al., TGF-beta1 induced transdifferentiation of rpe cells is mediated by TAK1, PLoS One 10 (4) (2015) e0122229. [11] X. Cang, et al., Mapping the functional binding sites of cholesterol in beta2adrenergic receptor by long-time molecular dynamics simulations, J. Phys. Chem. B 117 (4) (2013) 1085–1094.
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