Role of delta-like ligand-4 in chemoresistance against docetaxel in MCF-7 cells

Abstract

As Notch receptors have been shown to induce chemoresistance, we hypothesized that delta-like ligand-4 (DLL4), a central Notch signalling ligand, might also participate in chemoresistance in breast cancer. To investigate this issue, overexpression of DLL4 was induced by transfection with expression vectors for DLL4 in the human breast cancer cell line Michigan cancer foundation-7 (MCF-7). It was found that DLL4 could be adaptively upregulated by docetaxel (DOC) treatment in a dose-dependent manner, but Notch1 was unaffected. Overexpression of DLL4 could significantly attenuate the cytotoxic effects of DOC by increasing Bcl-2 expression, while decreasing Bax expression, apoptosis rate and DNA damage. The protective effects of DLL4 made cells acquire chemoresistance against DOC and resulted in cancer cell survival. DLL4 is normally regarded as a regulator of vascular development. Our results expanded the understanding of DLL4. Since DLL4 may play an important role in the process of acquiring chemoresistance, it may be a promising target in overcoming chemoresistance in breast cancer.

Keywords

DLL4 Notch1 chemoresistance docetaxel MCF-7 cells

Introduction

Breast cancer is one of the most common malignant tumours in women and is second only to lung cancer as a cause of cancer-related deaths in the United States. Over the past few decades, mortality rates associated with breast cancer have declined, largely associated with increased awareness, earlier detection and more effective treatment.¹ Chemotherapy is an important approach in the treatment of breast cancer with several drugs, such as docetaxel (DOC), proven to effectively treat breast cancer in clinical trails.^2,3

DOC, a cytotoxic taxane, is an antimicrotubule agent that principally exerts its cytotoxic activity by disrupting the microtubular network that is essential for mitotic and interphase cellular functions. It simultaneously promotes and stabilizes microtubule assembly and prevents microtubule depolymerization, thereby inhibiting normal cell division. In clinical trails, DOC monotherapy shows therapeutic efficacy in the treatment of metastatic breast cancer and its overall tolerability profile is generally considered acceptable for the majority of patients.^4,5 Consequently, DOC has been widely used in the chemotherapy against breast cancer. However, chemoresistance remains the most important obstacle restricting the clinical application of DOC.⁶

Chemoresistance of DOC is a complex phenomenon, with multiple factors and mechanisms contributing to the drug resistance. The underlying mechanisms of resistance acquisition to chemotherapeutic agents are still poorly understood. Many studies focus on signalling pathways, including Notch signalling, that inhibit cell death and result in cell survival. The Notch pathway is an evolutionally conserved signalling pathway that has been implicated in a wide variety of processes, including cell-fate determination, tissue morphogenesis, cell differentiation, proliferation and death.⁷ In mammals, the Notch system consists of four single-pass transmembrane receptors (Notch1–Notch4) and at least five membrane-anchored ligands (Jagged1, Jagged2, DLL1, DLL3 and DLL4). Notch signalling activation is initiated by ligand–receptor binding between two adjacent cells.⁸

The Notch signalling pathway has been implicated in tumour development. In several solid tumours, deregulated expression of wild-type Notch receptors, ligands and targets have been identified. However, the Notch pathway could be either oncogenic or tumour suppressive depending on the tissue and organ site in which it is expressed.^9,10 In human breast cancer, abnormal Notch1 activation was reported in 20 breast carcinomas of various subtypes, and high-level expression of Notch1 and Jagged1 has been correlated with poor prognosis.^11,12 Many studies have indicated that the Notch signalling plays an oncogenic role mainly through its interaction with other signalling pathways in mammary tumorigenesis in breast cancers. The well-characterized pathways which have interactions with Notch signalling during the oncogenesis of breast cancer include Erb2, TGF-β and Wnt signalling pathways.¹³ In addition, it has been reported that the Notch signalling pathway contributes to drug resistance in cancer cells. Inhibition of Notch signalling by Notch siRNA prevents drug resistance and sensitizes breast cancer cells to chemotherapy.¹⁴ Conversely, overexpression of Notch1 in non-transformed breast epithelial cells induces a general resistance to apoptotic stimuli by activating PI3K/AKT pathway.¹⁵ Thus, targeting Notch is a promising therapy to prevent drug resistance in breast cancer.

The ligands which activate the Notch signalling pathway play an important role regulating Notch signalling pathway and downstream. DLL4 is a Notch ligand with strong expression pattern in the endothelium of tumour blood vessels, which is notably different from Jagged1. In addition, DLL4 expression has been found in arterial endothelium during mouse embryogenesis, such as in the endothelium of the umbilical artery and axial dorsal aorta as well as in the endothelium of blood vessels.¹⁶ As revealed by the studies, DLL4 expression in human adult vasculature is low, but is upregulated in the tumour vessels.^17
–19 In clinical breast cancer samples, DLL4 was expressed by intratumoural endothelial cells and cells of ductal carcinomas. High intensity of DLL4 expression in endothelial cells was a statistically significant prognostic factor, which might suggest that breast tumours with high DLL4 expression in the vasculature progressed more rapidly.²⁰ Thus, treatment with DLL4 inhibitors reduces pericyte coverage and increases vascular leakage in tumours. An increase of vascular leakiness connected with impaired vascular integrity may explain a rapid decrease in tumour perfusion.^21,22 These findings clearly provide a rationale for DLL4 targeting for improved treatment of cancer patients.

DLL4 has been a promising target in angiogenesis-based cancer therapy, but the role of DLL4 in chemoresistance is still unknown. Targeting Notch, the receptor in this signalling pathway, has been proven to effectively reduce the chemoresistance in chemotherapy and promote cancer cell death. However, whether DLL4, or other Notch ligands, would participate in this process still needs to be determined, in addition to unearthing the specific mechanisms behind this effect. Here, we found that DOC led to cell apoptosis and induced expression of DLL4 in human breast cancer Michigan cancer foundation-7 (MCF-7) cells. In addition, overexpression of DLL4 in cells significantly alleviated the cell toxicity of DOC and resulted in cancer cell survival, indicating that DLL4 is associated with chemoresistance of DOC.

Methods and materials

Chemical agents

DOC was purchased from Sigma (St Louis, Missouri, USA) and MCF-7 cell line was from Cell Bank of Chinese Academy of Science (Shanghai, China). Dulbecco’s modified Eagle’s medium (DMEM) culture medium and foetal bovine serum were purchased from GIBCO (Grand Island, New York, USA). Hoechst 33258 dye was from Sigma. Cell Counter Kit-8 (CCK-8) cell viability assay kit was from Promoter Biotechnology (Nanjing, China). The reagents of lipofectamine 2000 and opti-MEM were purchased from Life Technology. Primary antibodies, including DLL4, Notch1, Bax, Bcl-2 (CST, California, USA) and β-actin were used in the study (Santa Cruze, California, USA). Annexin V-FITC/propidium iodide (PI) apoptosis detection kit was from Multi-sciences (Hangzhou, China).

Cell culture

MCF-7 human breast cancer cells were purchased from the Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China). Cells were maintained in DMEM medium with 10% heat-inactivated foetal bovine serum (containing 100 U/ml penicillin and 100 U/ml streptomycin) and incubated at 37^oC with 5% carbon dioxide. Culture medium was renewed every 2 days. Cells were collected and seeded into new culture bottles every 3 days. After incubation for 24 h, the medium was changed with serum-free medium containing different concentrations of DOC (2, 10, 50, 250 and 1250 nM/L) dissolved in water for experimentation.

Plasmid transfection

A human full-length DLL4 cDNA fragment was amplified using a forward primer 5′-GGAATTCACCATGGCGGCAGCGTCC-3′ and a reverse primer 5′-CGGGATCCTTATACCTCCGTGGCAATGACAC-3′, verified by sequencing. The vector of pCMV6-XL6 (Origene, Beijing, China) and verified DLL4 cDNA were digested by EcoRI and Sal 1, then applied to gel extraction and ligation by T4 DNA ligase (Thermo Scientific, Waltham, Massachusetts, USA), and verified by sequencing.

Transient transfection of MCF-7 cells was carried out using Lipofectamine 2000 following the manufacturer’s instructions. For six-well plates, the MCF-7 cells, at approximately 80% confluence, were transfected with 2 μg pCMV-DLL4 or pCMV-Con for 24 h, and then treated with DOC, followed by cell viability analysis, Annexin V-FITC/PI double staining assay and caspase-3/7 activity detection or Western blot analysis.

Cell viability assay

The cell viability was assessed by the CCK-8 assay (Dojindo Laboratories, Japan) according to the manufacturer’s instructions. MCF-7 cells were seeded in a 96-well plate and left to attach overnight. After the indicated treatments, 10 μM CCK-8 solution dissolved by serum-free DMEM medium was added to each well of the plate and the cells were incubated for 1 h on 37°C, and the absorbance was quantified on an automated microplate reader (Bio-Tec, California, USA).

Caspase-3/7 activity assay

Caspase-3/7 activity was measured with commercial kits (AAT Bioquest, Sunnyvale, California, USA). Cells planted in 96-well plate received indicated treatments and reacted with DEVD-AMC substrate in the reaction buffer, then incubated at room temperature for 1 h in the dark. The cell plate was centrifuged at 800 r/min for 2 min, then measured by an automated reader at E_x/E_m = 350/450 nM. The results were normalized to the number of cells present.

Annexin V-FITC/PI double staining assay

DOC-induced apoptosis was determined by flow cytometry using the Annexin V-FITC Apoptosis Detection kit (Keygen, Nanjing, china) as described by the manufacturer. Briefly, MCF-7 cells (2.5 × 10⁵ cells/well) were seeded into 6-well plates. At the end of the treatment, the cells were collected by centrifugation at 1000 r/min for 5 min and washed twice with ice-cold phosphate-buffered saline. The cells were resuspended in 500 μL of binding buffer and then stained with Annexin V-FITC solution (5 μL) and PI solution (5 μL) for 15 min at room temperature in dark. The samples were then analysed by flow cytometry (Becton Dickinson, San Jose, California, USA). A total of 10,000 cells were analysed for each sample. The percentage distributions of early apoptotic (lower right quadrant) and late apoptotic (upper right quadrant) were calculated for comparison.

Reverse transcription PCR analysis

RNA was extracted from MCF-7 cells with Trizol reagent (Invitrogen, Carlsbad, California, USA) according to the manufacturer’s protocol and was quantified and quality tested. Approximately 2 μg of total RNA was extracted for cDNA synthesis using Ominiscript RT kit (Thermo Scientific) in accordance with the manufacturer’s instructions. Real-time polymerase chain reaction (PCR) was performed in 10 µL containing 100 nM primers purchased from SANGON (Shanghai, China) and SYBR Green PCR Master Mix (Invitrogen). Sequences of primers were as follows: DLL4 forward 5′-CCCTGGCAATGTACTTGTGAT-3′; DLL4 reverse 5′-TGG TGG GTG CAG TAG TTG AG-3′; Notch forward 5′-GCC TCA ACA TCC CCT ACA AGA-3′; Notch reverse 5′-CCA CGA AGA ACA GAA GCA CAA A-3′; GAPDH forward 5′-CTG ACT TCA ACA GCG ACA CC-3′ and GAPDH reverse 5′-TGC TGT AGC CAA ATT CGT TGT -3′. The PCR conditions were as follows: 95°C for 2 min, 40 cycles at 95°C for 15 s and 60°C for 60 s. Each mRNA expression was normalized against GAPDH mRNA expression using the comparative cycle threshold method. The identity and purity of the amplified product were checked through analysis of the melting curve carried out at the end of amplification.

Western blot analysis

Whole cell proteins were extracted using ice-cold RIPA buffer (Beyotime institute of biotechnology, Shanghai, China) containing a protein inhibitor cocktail (Roche, Germany). After 40 min incubation at 4°C, the lysates were centrifuged, and the supernatants were obtained. The protein concentrations were determined by BCA protein assay kit (Pierce, Rockford, Illinois, USA) as described by the manufacturer.

Equivalent amounts of protein were separated by 10% SDS-polyacrylamide gel electrophoresis and transferred to NC membranes (Millipore Co, Billerica, Massachusetts, USA). Then the membranes were blocked in 5% non-fat milk or 5% BSA in TBST (10 mM Tris–HCl (pH 7.6), 0.1% Tween-20) for 1 h at room temperature, followed by incubation with either one of the following primary antibodies including DLL4, Notch and β-actin at 4°C overnight. The immunoblots were then incubated with species-appropriate secondary antibody conjugated with horseradish peroxidase (Abbkine, California, USA) for 1 h at room temperature. The membranes were developed using an electrochemiluminescence kit (Pierce, Rockford, Illinois, USA), according to the manufacturer’s protocol. The signals were detected and the density of the immunoreactive bands was analysed through Image J version 1.41 (National Institutes of Health, USA).

Statistical analysis

All results were expressed as means ± SD. All experiments were performed independently a minimum of three times. Statistical analyses were performed with SPSS18.0 software. Means were compared using one-way analysis of variance, and the least significant difference method was used to compare between groups. Significance was defined as p < 0.05.

Results

DLL4 overexpression improves the decreased cell viability induced by DOC in MCF-7 cells

To confirm the concentration relationship between DOC and cell viability in MCF-7 cells, we determined cell viability in MCF-7 treated with DOC using the CCK-8 assay. As shown in Figure 1(a), cells were treated with different concentrations of DOC (2, 10, 50, 250 and 1250 nmol/L) for 24 h. DOC showed clear cytotoxicity effects on MCF-7 cells in a dose-dependent manner. Cell viability decreased significantly after exposure of DOC at 50, 250 and 1250 nmol/L (p < 0.001). However, cell viability was noticeably increased after DLL4 plasmid transfection despite being treated with DOC for 24 h (Figure 1(b)). These results suggest DLL4 expression protected against DOC-induced cytotoxicity in MCF-7 cells.

Figure 1.

Overexpression of DLL4 attenuates DOC-induced cell death in MCF-7 cells. (a) Cells were treated with varied concentrations of DOC (2, 10, 50, 250 and 1250 nM) for 24 h, then subjected to CCK-8 analysis. (b) Similarly, cell viability was analysed in DOC-exposed cells pretreated with or without DLL4 plasmid transfection. The results are expressed as mean ± SD (n = 8). *p < 0.05, **p < 0.01, ***p < 0.001 versus control. DOC: docetaxel; DLL4: delta-like ligand-4; CCK-8: Cell Counter Kit-8.

DLL4 plasmid transfection enhances DLL4/Notch1 pathway activation caused by DOC in MCF-7 cells

To clarify whether DLL4/Notch1 signalling plays a role in DOC-induced toxicity, MCF-7 cells were treated with 10, 50 and 250 nmol/L DOC for 24 h in serum-free medium. Activation of DLL4/Notch1 was measured by Western blot analysis. DLL4 protein and mRNA expressions at concentrations of 50 and 250 nmol/L DOC were significantly increased (p < 0.05), whereas no significant changes in Notch1 protein and mRNA expressions were found (Figure 2(a) to (c)). This implies that DOC may activate the DLL4 ligand, yet not the Notch1 receptor. Hence, DLL4 plasmid transfection further increased DLL4 protein expression on the basis of DOC treatment (Figure 2(d)).

Figure 2.

DOC could activate DLL4/Notch1 pathway in MCF-7 cells. (a) Cells were treated with 10, 50 and 250 nM DOC for 24 h. Cell lysates were subjected to Western blot using antibodies against DLL4, Notch1 and β-actin. (b and c) The total mRNA ware extracted from MCF-7 cells treated with 10, 50 and 250 nM DOC for 12 h, the mRNA levels of DLL4 and Notch1 were quantified by reverse transcription polymerase chain reaction. (d) Proteins in cells treated with or without 50 nM DOC for 24 h after DLL4 or control plasmid transfection were detected by Western blot. Relative intensity was analysed by a laser scanning densitometer. The results were expressed as fold increases of optical density with β-actin serving as internal control. The results are expressed as mean ± SD (n = 3). *p < 0.05, **p < 0.01, ***p < 0.001 versus control; #p < 0.05, ##p < 0.01 versus pCMV-con + DOC. DOC: docetaxel; DLL4: delta-like ligand-4; CCK-8: Cell Counter Kit-8.

DLL4 overexpression affects DOC-induced Bcl-2/Bax expression in MCF-7 cells

In order to determine evidence of apoptosis, the expression of Bcl-2/Bax, a key member of the Bcl-2 protein family, was monitored. Treatment of MCF-7 cells with DOC (50 and 250 nmol/L) significantly downregulated Bcl-2 protein expression, whereas upregulated Bax protein expression compared to the control (p < 0.05) (Figure 3(a)). However, DLL4 overexpression increased markedly Bcl-2 protein level, and decreased significantly Bax protein level in MCF-7 cells exposed to DOC (p < 0.001; Figure 3(b)).

Figure 3.

DOC affects Bcl-2/Bax protein expression in MCF-7 cells. (a) The protein levels of Bcl-2 and Bax were detected in MCF-7 cells treated with 10, 50 and 250 nM DOC for 24 h. (b) After DLL4 or control plasmid transfection, cells were incubated with or without 50 nM DOC for 24 h, and Bcl-2/Bax protein levels were also determined by Western blot. The results were expressed as fold increases of optical density with β-actin serving as internal control. The results are expressed as mean ± SD (n = 3). *p < 0.05, **p < 0.01, ***p < 0.001 versus control; #p < 0.05, ##p < 0.01 versus pCMV-con + DOC. DOC: docetaxel; DLL4: delta-like ligand-4.

DLL4 plasmid transfection protects against DOC-induced apoptosis in MCF-7 cells

DNA fragmentation, a hallmark of apoptotic cells was assessed by Hoechst staining. Cells were treated with DOC for 24 h after DLL4 plasmid transfection to observe nuclear morphology. DOC at 50 nmol/L treatment caused condensation of cell bodies and nuclear fragmentation, whereas DLL4 overexpression alleviated relatively morphological changes associated with apoptosis (Figure 4(a)). The percentage of apoptotic cells was quantitated by FITC-annexin V/PI double staining with flow cytometry (Figure 4(b)). DOC at 50 nmol/L caused a 4.8-fold increase in apoptosis in MCF-7 cells after 24-h treatment compared to the control (p < 0.001). When cells were transfected with DLL4 plasmid, apoptosis induced by DOC was significantly decreased (p < 0.001). Furthermore, the activation of caspase-3/7 is regarded as hallmark of apoptosis. Caspase-3/7 activity was determined using a spectrophotometer. Caspase-3/7 activity significantly increased in MCF-7 cells treated with 10, 50 and 250 nmol/L DOC (p < 0.05; Figure 4(c)). As expected, caspase-3/7 activity significantly decreased in MCF-7 cells treated with 50 nmol/L DOC after DLL4 plasmid transfection (Figure 4(d)).

Figure 4.

DLL4 protects against DOC-induced apoptosis. MCF-7 cells pretreated with DLL4 or control plasmid transfection were exposed to 50 nM DOC for 24 h. (a) Nuclear morphology was visualized using Hoechst staining, scale bar: 50 μM. (b) Apoptosis in cells was statistically analysed by flow cytometry and expressed as a percentage. The results are expressed as mean ± SD (n = 4). (c) MCF-7 cells were treated with 10, 50 or 250 nM DOC, then the protein lysates were extracted and subjected to a caspase-3/7 activity analysis. Caspase-3/7 activity was expressed as arbitrary units relative to the vehicle control cells. (d) Caspased 3/7 activity analysis was performed in cells exposed with or without 50 nM DOC after plasmid transfection. The results are expressed as mean ± SD (n = 6). *p < 0.05, **p < 0.01, ***p <0.001 versus control; #p < 0.05, versus pCMV-con + DOC. DOC: docetaxel; DLL4: delta-like ligand-4.

Discussion

Initially in this study, apoptosis of MCF-7 cells induced by DOC was studied. DOC is active against a range of human cancers, including breast cancer. Its antitumour activity is mainly based on stabilization of microtubule dynamics and thereby disruption of the cell cycle and has also been shown to induce cell death (by apoptosis or cell lysis) in many tumour cell lines.^23,24 Many studies have certified that DOC could induce cell caspase-3 activation,²⁵ decrease the expression of the anti-apoptotic protein Bcl-2 and increase the pro-apoptosis protein Bax²⁶ while also causing DNA damage.²⁷ It is well known that Bcl-2 is related to survival pathways involving the mitochondrial membrane of cancer cells, thus contributing to chemotherapy resistance. On the contrary, Bax plays a pro-apoptotic role in cells. The value of Bcl-2/Bax ratio is an important indicator for regulation of apoptosis.^28,29 In this study, we observed increased apoptosis rate and decreased cell viability in MCF-7 cells treated with varied dose of DOC. In addition, DOC activated caspase-3/7 activity, upregulated Bax and downregulated Bcl-2 in a dose-dependent manner. In essence, DOC could efficiently kill MCF-7 breast cancer cells.

The induction of apoptosis in cancer cells is an important part of antitumour effects of DOC. However, chemoresistance presents a major obstacle to cell death effects of DOC treatment as cancer cell survival is a result of this resistance. Doxorubicin-resistant MCF-7 cells are 10 times more resistant to DOC than the sensitive wild-type cell line and the occurrence of apoptosis has not been readily observed.³⁰ DLL4/Notch1 signalling pathway has been proven to be associated with breast cancer. Notch receptor expression levels have been found to be elevated in human breast cancer cell lines and specimens. However, in most studies regarding human breast cancers, activated Notch is only detectable at the protein level, rather than the mRNA level.^11,31 In the present study, we also found that DOC induced DLL4 transcription and expression in a dose-dependent manner, yet scarcely affected the expression of Notch1 in MCF-7 cells. The results suggested that the activation of DLL4/Notch1 signal pathway was mainly dependent on DLL4 elevation. In fact, DLL4 could bind to Notch receptors and thereby activate Notch signalling pathways. Several studies have found that knockdown or knockout of Notch1 to inhibit Notch signalling pathway could successfully lead to cell growth inhibition and enhance chemosensitivity in human breast cancer.¹⁴ In the present study, the adaptive activation of Notch signalling through DLL4 elevation during DOC treatment indicated that the ligands of Notch signalling might also participate in the process.

To understand the role of DLL4 mediation of the Notch signal pathway activation in DOC therapy against breast cancer, overexpression of DLL4 was applied in this study. The results confirmed that elevated DLL4 in MCF-7 cells significantly precluded the antitumour effects of DOC which led to cancer cell death. Cells which overexpressed DLL4 exhibited lower caspase-3/7 activity, reduced DNA damage, increased Bcl2 expression as well as decreased Bax expression compared with the control group. Therefore, cytotoxicity of DOC in breast cancer cells was inhibited and the cells exhibited chemoresistence against DOC.

Cancer cells that acquire resistance to chemotherapeutic agent exert a considerable survival advantage. This occurs mainly by activating survival pathways, or by inhibiting apoptotic pathways, of which the Notch signalling pathway is a key regulator through mechanisms that may be similar to its role in tumorigenesis. Treatment of colorectal cancer with oxaliplatin activates the Notch pathway and pro-survival pathways, such as PI3K/Akt pathway. Moreover, blocking Notch activation sensitizes these cells to chemotherapeutic drugs.³² In pancreatic cancer, the expression of Notch3 along with phospho-STAT3 and phospho-Akt is associated with an aggressive tumour phenotype.³³ Inhibition of the PI3K survival pathway with wortmannin or LY294002 results in reduced levels of the Notch intracellular domain in prostate cancer cells. This leads to loss of Notch-mediated p53 downregulation and thus increases the cells susceptibility to chemotherapeutic agents.³⁴ Similar effects have been observed by blocking mTOR with rapamycin, which prevents the inhibition of p53-mediated transcription by Notch, thus sensitizing the cells to drug treatment.³⁵ Notch-induced chemoresistance can also result from antagonism between Notch and epidermal growth factor receptor, as observed in trastuzumab-resistant ERBB2-positive breast cancer.³⁶ In the maintenance of ER-negative breast tumours, an important role has been attributed to Notch. Tumours exerting high level of Notch show an increased expression of survivin, increased cell proliferation and reduced apoptosis.^37,38 These evidences indicate that the activation of Notch pathway could make tumour cells resistant to chemotherapy or radiation. In this study, we proved that DLL4, one ligand of Notch receptors, participated in the acquiring chemoresistance against DOC in breast cancer line MCF-7.

As a ligand of Notch signal pathway, DLL4 has the ability to activate Notch signal pathway and thus participate in wide range of cellular processes, including survival pathways and apoptotic pathways.^13,39 In fact, DLL4 inhibitor or anti-DLL4 antibodies have been demonstrated as a promising target in angiogenesis-based cancer therapy.^22,40 In clinical practice, angiogenesis inhibition by way of vascular endothelial growth factor (VEGF) blockade is demonstrated to produce a great number of benefits.^41,42 Nevertheless, resistance to VEGF inhibitors is emerging as a significant problem of clinical oncology.¹⁸ Fortunately, some studies have found that blockade of DLL4 results in non-productive angiogenesis with an inhibitory effect on tumour growth, including tumour resisted to VEGF inhibitors.¹⁷ Moreover, blocking DLL4 signalling has an additive or synergistic effect on delaying tumour growth when used in combination with anti-VEGF treatments.⁴³ Interestingly, in some tumour varieties, overexpression of DLL4 in tumour cells does not correlate with increased tumour growth in vivo.^44,45 This indicates that overexpression of DLL4 may make cancer cells acquire survival advantage through Notch signal pathway activation, as discussed previously.

Taking into consideration the results of this study, we found that DOC treatment caused DLL4 transcription and expression as an adaptive response but not Notch1. In addition, upregulation of DLL4 could impede DOC-induced cell apoptosis in breast cancer cell line MCF-7. These findings indicate that though the activation of Notch signalling pathway by increased level of Notch receptors which have been demonstrated participating in chemoresistance, DLL4, the ligands, might also play an important role in acquiring chemoresistance to DOC. Our study expands the understanding of the role of DLL4/Notch1 signalling pathway in chemoresistance in breast cancer, providing the potential application of therapy of targeting DLL4 to overcome chemoresistance to DOC.

Footnotes

Declaration of Conflicting Interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This work was financially supported by Science and Technology Research Project of Hubei Provincial Department of Education (no. Q20132107).

References

Gradishar

Anderson

Blair

. Breast cancer version 3.2014. J Natl Compr Canc Netw 2014; 12: 542–590.

Jones

Erban

Overmoyer

. Randomized phase III study of docetaxel compared with paclitaxel in metastatic breast cancer. J Clin Oncol 2005; 23: 5542–5551.

Nabholtz

Senn

Bezwoda

. Prospective randomized trial of docetaxel versus mitomycin plus vinblastine in patients with metastatic breast cancer progressing despite previous anthracycline-containing chemotherapy. 304 Study Group. J Clin Oncol 1999; 17: 1413–1424.

Dieras

Girre

Pierga

. Update on docetaxel and breast cancer. Bull Cancer 2004; 91: 409–417.

Lyseng-Williamson

Fenton

. Docetaxel: a review of its use in metastatic breast cancer. Drugs 2005; 65: 2513–2531.

Feng

Wang

Song

. MicroRNA-200b reverses chemoresistance of docetaxel-resistant human lung adenocarcinoma cells by targeting E2F3. Cancer 2012; 118: 3365–3376.

Bray

. Notch signalling: a simple pathway becomes complex. Nat Rev Mol Cell Biol 2006; 7: 678–689.

Mumm

Schroeter

Saxena

. A ligand-induced extracellular cleavage regulates gamma-secretase-like proteolytic activation of Notch1. Mol Cell 2000; 5: 197–206.

Reedijk

. Notch signaling and breast cancer. Adv Exp Med Biol 2012; 727: 241–257.

10.

Roy

Pear

Aster

. The multifaceted role of Notch in cancer. Curr Opin Genet Dev 2007; 17: 52–59.

11.

Reedijk

Odorcic

Chang

. High-level coexpression of JAG1 and NOTCH1 is observed in human breast cancer and is associated with poor overall survival. Cancer Res 2005; 65: 8530–8537.

12.

Stylianou

Clarke

Brennan

. Aberrant activation of notch signaling in human breast cancer. Cancer Res 2006; 66: 1517–1525.

13.

Yin

Velazquez

Liu

. Notch signaling: emerging molecular targets for cancer therapy. Biochem Pharmacol 2010; 80: 690–701.

14.

Zang

Chen

Dai

. RNAi-mediated knockdown of Notch-1 leads to cell growth inhibition and enhanced chemosensitivity in human breast cancer. Oncol Rep 2010; 23: 893–899.

15.

Meurette

Stylianou

Rock

. Notch activation induces Akt signaling via an autocrine loop to prevent apoptosis in breast epithelial cells. Cancer Res 2009; 69: 5015–5022.

16.

Mailhos

Modlich

Lewis

. Delta4, an endothelial specific notch ligand expressed at sites of physiological and tumor angiogenesis. Differentiation 2001; 69: 135–144.

17.

Sainson

Harris

. Anti-Dll4 therapy: can we block tumour growth by increasing angiogenesis? Trends Mol Med 2007; 13: 389–395.

18.

Sainson

Shi

. Delta-like 4 Notch ligand regulates tumor angiogenesis, improves tumor vascular function, and promotes tumor growth in vivo. Cancer Res 2007; 67: 11244–11253.

19.

Sainson

Oon

. DLL4-Notch signaling mediates tumor resistance to anti-VEGF therapy in vivo. Cancer Res 2011; 71: 6073–6083.

20.

Jubb

Soilleux

Turley

. Expression of vascular notch ligand delta-like 4 and inflammatory markers in breast cancer. Am J Pathol 2010; 176: 2019–2028.

21.

Kuhnert

Kirshner

Thurston

. Dll4-Notch signaling as a therapeutic target in tumor angiogenesis. Vasc Cell 2011; 3: 20.

22.

Brzozowa

Wojnicz

Kowalczyk-Ziomek

. The Notch ligand Delta-like 4 (DLL4) as a target in angiogenesis-based cancer therapy? Contemp Oncol (Pozn) 2013; 17: 234–237.

23.

Figgitt

Wiseman

. Docetaxel: an update of its use in advanced breast cancer. Drugs 2000; 59: 621–651.

24.

Hortobagyi

. Recent progress in the clinical development of docetaxel (Taxotere). Semin Oncol 1999; 26: 32–36.

25.

Colarossi

Memeo

Colarossi

. Inhibition of histone deacetylase 4 increases cytotoxicity of docetaxel in gastric cancer cells. Proteomics Clin Appl 2014; 8: 924–931.

26.

Zhang

Peng

. Silencing Notch-1 induces apoptosis and increases the chemosensitivity of prostate cancer cells to docetaxel through Bcl-2 and Bax. Oncol Lett 2012; 3: 879–884.

27.

Iida

Shimada

Sakagami

. Cytotoxicity induced by docetaxel in human oral squamous cell carcinoma cell lines. In Vivo 2013; 27: 321–332.

28.

Adams

Cory

. The Bcl-2 apoptotic switch in cancer development and therapy. Oncogene 2007; 26: 1324–1337.

29.

Driak

Dvorska

Svandova

. Changes in expression of some apoptotic markers in different types of human endometrium. Folia Biol (Praha) 2011; 57: 104–111.

30.

Wang

Hajar

. Multiple mechanisms underlying acquired resistance to taxanes in selected docetaxel-resistant MCF-7 breast cancer cells. BMC Cancer 2014; 14: 37.

31.

Parr

Watkins

Jiang

. The possible correlation of Notch-1 and Notch-2 with clinical outcome and tumour clinicopathological parameters in human breast cancer. Int J Mol Med 2004; 14: 779–786.

32.

Meng

Shelton

. Gamma-secretase inhibitors abrogate oxaliplatin-induced activation of the Notch-1 signaling pathway in colon cancer cells resulting in enhanced chemosensitivity. Cancer Res 2009; 69: 573–582.

33.

Doucas

Mann

Sutton

. Expression of nuclear Notch3 in pancreatic adenocarcinomas is associated with adverse clinical features, and correlates with the expression of STAT3 and phosphorylated Akt. J Surg Oncol 2008; 97: 63–68.

34.

Wang

Banerjee

. Down-regulation of Notch-1 and Jagged-1 inhibits prostate cancer cell growth, migration and invasion, and induces apoptosis via inactivation of Akt, mTOR, and NF-kappaB signaling pathways. J Cell Biochem 2010; 109: 726–736.

35.

Mungamuri

Yang

Thor

. Survival signaling by Notch1: mammalian target of rapamycin (mTOR)-dependent inhibition of p53. Cancer Res 2006; 66: 4715–4724.

36.

Rousseau

Nichol

Pettersson

. ERbeta sensitizes breast cancer cells to retinoic acid: evidence of transcriptional crosstalk. Mol Cancer Res 2004; 2: 523–531.

37.

Lee

Simin

Liu

. A functional Notch-survivin gene signature in basal breast cancer. Breast Cancer Res 2008; 10: R97.

38.

Lee

Raskett

Prudovsky

. Molecular dependence of estrogen receptor-negative breast cancer on a notch-survivin signaling axis. Cancer Res 2008; 68: 5273–5281.

39.

Billiard

Kirshner

Tait

. Ongoing Dll4-Notch signaling is required for T-cell homeostasis in the adult thymus. Eur J Immunol 2011; 41: 2207–2216.

40.

Ridgway

Zhang

. Inhibition of Dll4 signalling inhibits tumour growth by deregulating angiogenesis. Nature 2006; 444: 1083–1087.

41.

Verheul

Pinedo

. Possible molecular mechanisms involved in the toxicity of angiogenesis inhibition. Nat Rev Cancer 2007; 7: 475–485.

42.

Bagnasco

Piras

Parodi

. Role of angiogenesis inhibitors in colorectal cancer: sensitive and insensitive tumors. Curr Cancer Drug Targets 2012; 12: 303–315.

43.

Thurston

Noguera-Troise

Yancopoulos

. The Delta paradox: DLL4 blockade leads to more tumour vessels but less tumour growth. Nat Rev Cancer 2007; 7: 327–331.

44.

Scehnet

Jiang

Kumar

. Inhibition of Dll4-mediated signaling induces proliferation of immature vessels and results in poor tissue perfusion. Blood 2007; 109: 4753–4760.

45.

Noguera-Troise

Daly

Papadopoulos

. Blockade of Dll4 inhibits tumour growth by promoting non-productive angiogenesis. Novartis Found Symp 2007; 283: 106–120; discussion 121–105, 238–141.